CN110088133B - Anti-idiotype antibodies and related methods - Google Patents

Anti-idiotype antibodies and related methods Download PDF

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CN110088133B
CN110088133B CN201780059515.0A CN201780059515A CN110088133B CN 110088133 B CN110088133 B CN 110088133B CN 201780059515 A CN201780059515 A CN 201780059515A CN 110088133 B CN110088133 B CN 110088133B
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antibody
antigen
cdr
binding fragment
seq
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CN110088133A (en
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C·豪金斯
M·D·亨佩尔
C·L·萨瑟兰
T·萨哈利亚
J·史密斯
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Juno Therapeutics Inc
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Juno Therapeutics Inc
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/42—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against immunoglobulins
    • C07K16/4208—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against immunoglobulins against an idiotypic determinant on Ig
    • C07K16/4241—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against immunoglobulins against an idiotypic determinant on Ig against anti-human or anti-animal Ig
    • C07K16/4258—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against immunoglobulins against an idiotypic determinant on Ig against anti-human or anti-animal Ig against anti-receptor Ig
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/42—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against immunoglobulins
    • C07K16/4208—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against immunoglobulins against an idiotypic determinant on Ig
    • C07K16/4241—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against immunoglobulins against an idiotypic determinant on Ig against anti-human or anti-animal Ig
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0636—T lymphocytes
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6854—Immunoglobulins
    • G01N33/686—Anti-idiotype
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/622—Single chain antibody (scFv)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/75—Agonist effect on antigen
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/03—Fusion polypeptide containing a localisation/targetting motif containing a transmembrane segment
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/33—Fusion polypeptide fusions for targeting to specific cell types, e.g. tissue specific targeting, targeting of a bacterial subspecies
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/50—Cell markers; Cell surface determinants
    • C12N2501/599—Cell markers; Cell surface determinants with CD designations not provided for elsewhere

Abstract

Provided herein are anti-idiotype antibodies that specifically recognize anti-CD 19 antibody moieties, particularly anti-CD 19 antibody moieties present in recombinant receptors, including Chimeric Antigen Receptors (CARs). The disclosure further relates to the use of an anti-idiotype antibody for specifically identifying and/or selecting cells expressing such recombinant receptors, such as anti-CD 19CAR T cells. The disclosure further relates to the use of an anti-idiotype antibody for specifically activating such cells.

Description

Anti-idiotype antibodies and related methods
Cross Reference to Related Applications
The present application claims the benefit of priority from U.S. provisional patent application 62/369,008 entitled "antibody and related methods" filed on 7/29 of 2016, the contents of which are incorporated herein by reference in their entirety for all purposes.
By introduction into the sequence listing
The present application is presented with a sequence listing in electronic format. The sequence listing provided is a file created on 2017, 7, 15, named 735042006540seqlist.txt, of size 86,050 bytes. The electronic format information of the sequence listing is fully incorporated by reference.
FIELD
The present disclosure relates in some aspects to anti-idiotype antibodies that specifically recognize anti-CD 19 antibody moieties, particularly anti-CD 19 antibody moieties present in recombinant receptors, including Chimeric Antigen Receptors (CARs). The disclosure further relates to the use of an anti-idiotype antibody for specifically identifying or selecting cells expressing such recombinant receptors, such as anti-CD 19CAR T cells. The disclosure further relates to the use of an anti-idiotype antibody for specifically activating such cells.
Background
Methods using engineered cells expressing recombinant receptors, such as Chimeric Antigen Receptors (CARs) containing extracellular antibody antigen binding domains, can be used for adoptive cell therapy. There are various strategies available for assessing the activity of such cells in vitro or in vivo in a subject. There is a need for improved methods to specifically evaluate the activity of CAR-expressing cells. Reagents, compositions and articles of manufacture are provided that meet this need.
SUMMARY
Provided herein are agents, including antibody fragments (such as scFv) and chimeric molecules (such as chimeric antigen receptors) containing them, that specifically bind to antibodies. Compositions and articles of manufacture comprising such agents, including those having a surface (such as a solid surface) to which the agents are bound, such as plates or beads, are also provided. In embodiments, also provided herein are uses and methods of use of such agents, compositions and articles of manufacture, including for detecting, using, manipulating and/or stimulating cells or therapies containing or suspected of containing an antibody or chimeric molecule, such as for detecting, stimulating or using cells expressing a CAR.
In some aspects, the antibody is or comprises an anti-idiotype antibody or antigen-binding fragment thereof that specifically binds to a target antibody that is or comprises the variable region of an antibody designated SJ25C1, and/or an antigen-binding fragment thereof. In some embodiments, the agent (e.g., an anti-idiotype antibody or antigen-binding fragment) comprises a light chain Variable (VL) region having at least 90% sequence identity (and/or at least 95% or 99% sequence identity, or 100% identity) to the VL region amino acid sequence set forth in SEQ ID No. 5; and/or a heavy chain Variable (VH) region having at least 90% sequence identity (and/or at least 95% or 99% sequence identity, or 100% identity) to the VH region amino acid sequence set forth in SEQ ID No. 1.
Provided herein are antibodies or antigen-binding fragments thereof, wherein the antibodies or antigen-binding fragments comprise a VL region having at least 90% sequence identity (and/or at least 95% or 99% sequence identity, or 100% identity) to the VL region amino acid sequence set forth in SEQ ID No. 5; and/or a VH region having at least 90% sequence identity (and/or at least 95% or 99% sequence identity, or 100% identity) to the VH region amino acid sequence set forth in SEQ ID No. 1.
In some of any such embodiments, the VH region comprises heavy chain complementarity determining region 3 (CDR-H3), said CDR-H3 comprising the amino acid sequence set forth in SEQ ID No. 11 or 84 or comprising CDR-H3 comprised within the VH sequence set forth in SEQ ID No. 1; and/or the VL region comprises light chain complementarity determining region 3 (CDR-L3), said CDR-L3 comprising the amino acid sequence set forth in SEQ ID NO. 14 or 87 or comprising CDR-L3 comprised within the VL sequence set forth in SEQ ID NO. 5.
In some of any such embodiments, the VH region contains CDR-H1 and CDR-H2, which contain the amino acid sequences of CDR-H1 and CDR-H2 sequences contained within the amino acid sequences of the VH region set forth in SEQ ID NO 1, respectively; and/or the VL region comprises a CDR-L1 and a CDR-L2 comprising the amino acid sequences of the CDR-L1 and CDR-L2 sequences, respectively, contained within the amino acid sequences of the VL region set forth in SEQ ID NO. 5.
In some of any such embodiments, the VH region contains the CDR-H1 set forth in SEQ ID NO 9, 78, 79 or 80, the CDR-H2 set forth in SEQ ID NO 10, 81, 82 or 83, and the CDR-H3 set forth in SEQ ID NO 11 or 84; and/or the VL region comprises the CDR-L1 set forth in SEQ ID NO. 12 or 85, the CDR-L2 set forth in SEQ ID NO. 13 or 86, and the CDR-L3 set forth in SEQ ID NO. 14 or 87.
Provided herein are anti-idiotype antibodies or antigen-binding fragments thereof comprising CDR-H1, CDR-H2 and CDR-H3 and/or CDR-L1, CDR-L2 and CDR-L3, said CDR-H1, CDR-H2 and CDR-H3 comprising the amino acid sequences of CDR-H1, CDR-H2 and CDR-H3 sequences, respectively, comprised within the amino acid sequences of the VH regions set forth in SEQ ID No. 1; the CDR-L1, CDR-L2 and CDR-L3 contain the amino acid sequences of CDR-L1, CDR-L2 and CDR-L3 sequences, respectively, contained within the amino acid sequences of the VL region set forth in SEQ ID NO. 5.
Provided herein are anti-idiotype antibodies or antigen-binding fragments thereof, comprising a CDR-H1 comprising the amino acid sequence of SEQ ID No. 9, 78, 79 or 80, a CDR-H2 comprising the amino acid sequence of SEQ ID No. 10, 81, 82 or 83, and a CDR-H3 comprising the amino acid sequence as set forth in SEQ ID No. 11 or 84; and/or CDR-L1 comprising the amino acid sequence SEQ ID NO. 12 or 85, CDR-L2 comprising the amino acid sequence SEQ ID NO. 13 or 86, and CDR-L3 comprising the amino acid sequence SEQ ID NO. 14 or 87.
In some of any such embodiments, the VH region of the antibody or fragment contains the amino acid sequence of SEQ ID No. 1 and/or the VL region of the antibody or fragment contains the amino acid sequence of SEQ ID No. 5. In some embodiments, the VH region of the antibody or fragment contains the amino acid sequence SEQ ID NO. 1 and the VL region of the antibody or fragment contains the amino acid sequence SEQ ID NO. 5. In some of any such embodiments, the target antibody or antigen-binding fragment contains the heavy chain variable region set forth in SEQ ID NO. 23 and/or the light chain variable region set forth in SEQ ID NO. 24.
In some embodiments, the agent is an anti-idiotype antibody or antigen-binding fragment thereof that specifically binds to a target antibody (which is or contains the variable region of an antibody designated FMC 63) or antigen-binding fragment thereof and/or to a chimeric molecule containing such an antibody fragment, such as a CAR having a binding domain containing an antibody variable region derived from FMC63 or a portion thereof (such as in scFv format). In some embodiments, the agent (e.g., an anti-idiotype antibody or antigen-binding fragment) comprises a light chain Variable (VL) region having at least 90% sequence identity to the VL region amino acid sequence set forth in SEQ ID No. 40 or 62; and/or a heavy chain Variable (VH) region having at least 90% sequence identity (and/or at least 95% or 99% sequence identity, or 100% identity) to the VH region amino acid sequence set forth in SEQ ID No. 36 or 58. In some embodiments, the antibody or antigen binding fragment contains a VL region that has at least 90% sequence identity (and/or at least 95% or 99% sequence identity, or 100% identity) to the VL region amino acid sequence set forth in SEQ ID nos. 40 or 62; and/or a VH region having at least 90% sequence identity (and/or at least 95% or 99% sequence identity, or 100% identity) to the VH region amino acid sequence set forth in SEQ ID No. 36 or 58.
In some of any such embodiments, the VH region contains a sequence comprising amino acid sequence GYX 3 FX 5 X 6 YX 8 MX 10 Heavy chain complementarity determining region 1 (CDR-H1) (SEQ ID NO: 108), wherein X 3 Is T or S, X 5 Is T or S, X 6 Is D or R, X 8 Is Y or W, and X 10 Is K or N; comprising the amino acid sequence WIGX 4 IX 6 PX 8 X 9 X 10 X 11 TX 13 X 14 NQX 17 FKX 20 Heavy chain complementarity determining region 2 (CDR-H2) of (SEQ ID NO: 109), wherein X 4 Is D or M, X 6 Is N or H, X 8 Is N or S, X 9 Is N or D, X 10 Is G or S, X 11 Is G or E, X 13 Is D or R, X 14 Y or L, X 17 Is N or K, and X 20 Is G or D; comprising the amino acid sequence AX 2 X 3 X 4 X 5 X 6 X 7 X 8 X 9 X 10 X 11 X 1 2 X 13 X 14 X 15 Heavy chain complementarity determining region 3 (CDR-H3) of (SEQ ID NO: 110), wherein X 2 Is R or S, X 3 Is E or I, X 4 Is G or Y, X 5 Is N or Y, X 6 Is N or E, X 7 Is Y or null, X 8 Is G or null, X 9 Is S or null, X 10 R is null or null, X 11 Is D or null, X 12 Is A or null value, X 13 Is M or null, X 14 Is D or E, and X 15 Y or A; and/or the VL region comprises an amino acid sequence X 1 AX 3 X 4 X 5 X 6 X 7 X 8 YX 10 X 11 Light chain complementarity determining region 1 (CDR-L1) of WY (SEQ ID NO: 111), wherein X 1 Is S or R, X 3 Is S or R, X 4 Is S or G, X 5 Is G or N, X 6 Is V or I, X 7 Is I or H, X 8 Is N or null, X 10 Is M or L, and X 11 Y or A; comprising the amino acid sequence X 1 X 2 X 3 YX 5 X 6 X 7 X 8 LAX 11 Light chain complementarity determining region 2 (CDR-L2) of (SEQ ID NO: 112), wherein X 1 Is P or L, X 2 Is W or L, X 3 Is I or V, X 5 Is L or N, X 6 Is T or A, X 7 Is S or K, X 8 Is N or T, and X 11 Is S or D; comprising the amino acid sequence QX 2 X 3 X 4 X 5 X 6 PX 8 T (SEQ ID NO: 113) light chain complementarity determining region 3 (CDR-L3), wherein X 2 Is Q or H, X 3 Is W or F, X 4 Is S or W, X 5 Is S or W, X 6 Is N or T, and X 8 Is L or Y.
In some embodiments, complementarity determining region 3 (CDR-H3) contains the amino acid sequence set forth in SEQ ID NO. 94 or 104 or contains CDR-H3 contained within the VH sequence set forth in SEQ ID NO. 36 or 58; and/or light chain complementarity determining region 3 (CDR-L3) contains the amino acid sequence set forth in SEQ ID NO. 97 or 107 or contains CDR-L3 contained within the VL sequence set forth in SEQ ID NO. 40 or 62.
In some embodiments, the VH region contains CDR-H1 and CDR-H2, which include the amino acid sequences of CDR-H1 and CDR-H2 sequences contained within the amino acid sequences of the VH region set forth in SEQ ID NO 36 or 58, respectively; and/or the VL region comprises CDR-L1 and CDR-L2, which comprise the amino acid sequences of CDR-L1 and CDR-L2 sequences contained within the amino acid sequences of the VL region set forth in SEQ ID NO. 40 or 62, respectively.
In some of any such embodiments, the VH region contains the CDR-H1 set forth in SEQ ID NO 88, 89, 90, 98, 99 or 100, the CDR-H2 set forth in SEQ ID NO 91, 92, 93, 101, 102 or 103, and the CDR-H3 set forth in SEQ ID NO 94 or 104; and/or the VL region comprises CDR-L1 as set forth in SEQ ID NO 95 or 105, CDR-L2 as set forth in SEQ ID NO 96 or 106, and CDR-L3 as set forth in SEQ ID NO 97 or 107.
Provided herein are anti-idiotype antibodies or antigen-binding fragments thereof comprising CDR-H1, CDR-H2 and CDR-H3 and/or CDR-L1, CDR-L2 and CDR-L3, said CDR-H1, CDR-H2 and CDR-H3 comprising the amino acid sequences of CDR-H1, CDR-H2 and CDR-H3 sequences comprised within the amino acid sequences of the VH regions set forth in SEQ ID NOs 36 or 58, respectively; the CDR-L1, CDR-L2 and CDR-L3 include the amino acid sequences of CDR-L1, CDR-L2 and CDR-L3 sequences contained within the amino acid sequences of the VL region set forth in SEQ ID NO. 40 or 62, respectively.
Provided herein are anti-idiotype antibodies, or antigen-binding fragments thereof, comprising CDR-H1 comprising amino acid sequence SEQ ID NOs 88, 89, 90, 98, 99 or 100, CDR-H2 comprising amino acid sequence SEQ ID NOs 91, 92, 93, 101, 102 or 103, and CDR-H3 comprising amino acid sequences as set forth in SEQ ID NOs 94 or 104; and/or CDR-L1 comprising amino acid sequence SEQ ID NO 95 or 105, CDR-L2 comprising amino acid sequence SEQ ID NO 96 or 106, and CDR-L3 comprising amino acid sequence SEQ ID NO 97 or 107.
In some of any such embodiments, the VH region of the antibody or fragment contains the amino acid sequence SEQ ID NO 36 or 58 and/or the VL region of the antibody or fragment contains the amino acid sequence SEQ ID NO 40 or 62. In some cases, the VH region of the antibody or fragment comprises the amino acid sequence SEQ ID NO 36 or 58 and the VL region of the antibody or fragment comprises the amino acid sequence SEQ ID NO 40 or 62.
In some of any such embodiments, the VH region contains the CDR-H1 set forth in SEQ ID NO 44, 88, 89 or 90, the CDR-H2 set forth in SEQ ID NO 45, 91, 92 or 93 and the CDR-H3 set forth in SEQ ID NO 46 or 94; and/or the VL region comprises CDR-L1 as set forth in SEQ ID NO. 47 or 95, CDR-L2 as set forth in SEQ ID NO. 48 or 96, and CDR-L3 as set forth in SEQ ID NO. 49 or 97. In some of any such embodiments, the VH region contains the CDR-H1 set forth in SEQ ID NO 65, 98, 99 or 100, the CDR-H2 set forth in SEQ ID NO 66, 101, 102 or 103 and the CDR-H3 set forth in SEQ ID NO 67 or 104; and/or the VL region comprises CDR-L1 as set forth in SEQ ID NO 68 or 105, CDR-L2 as set forth in SEQ ID NO 69 or 106, and CDR-L3 as set forth in SEQ ID NO 100 or 107.
In some of any such embodiments, the VH region contains CDR-H1, CDR-H2 and CDR-H3, which include the amino acid sequences of CDR-H1, CDR-H2 and CDR-H3 sequences contained within the amino acid sequences of the VH region set forth in SEQ ID NO:36, respectively; and/or the VL region comprises CDR-L1, CDR-L2 and CDR-L3, which comprise the amino acid sequences of CDR-L1, CDR-L2 and CDR-L3 sequences, respectively, contained within the amino acid sequences of the VL region set forth in SEQ ID NO. 40.
In some of any such embodiments, the VH region contains CDR-H1, CDR-H2 and CDR-H3, which include the amino acid sequences of CDR-H1, CDR-H2 and CDR-H3 sequences contained within the amino acid sequence of the VH region set forth in SEQ ID NO 58, respectively; and/or the VL region comprises CDR-L1, CDR-L2 and CDR-L3, which comprise the amino acid sequences of CDR-L1, CDR-L2 and CDR-L3 sequences, respectively, contained within the amino acid sequences of the VL region set forth in SEQ ID NO. 62.
In some of any such embodiments, the VH region of the antibody or fragment comprises the amino acid sequence SEQ ID NO:36 and/or the VL region of the antibody or fragment comprises the amino acid sequence SEQ ID NO:40. In some of any such embodiments, the VH region of the antibody or fragment comprises the amino acid sequence SEQ ID NO:58 and/or the VL region of the antibody or fragment comprises the amino acid sequence SEQ ID NO:62.
In some of any such embodiments, the target antibody or antigen-binding fragment is a single-chain fragment. In some aspects, the fragments contain antibody variable regions linked by flexible linkers. In some of any such embodiments, the fragment contains an scFv.
In some of any such embodiments, the target antibody or antigen-binding fragment contains the heavy chain variable region set forth in SEQ ID NO. 23 and/or the light chain variable region set forth in SEQ ID NO. 24; and/or is an scFv comprising the amino acid sequence set forth in SEQ ID NO. 28. In some embodiments, the target antibody or antigen-binding fragment contains the heavy chain variable region set forth in SEQ ID NO. 30 and/or the light chain variable region set forth in SEQ ID NO. 31; and/or is an scFv comprising the amino acid sequence set forth in SEQ ID NO 34.
In some of any of such embodiments, the anti-idiotype antibody or antigen-binding fragment specifically binds to an epitope of the target antibody or antigen-binding fragment thereof that is the same as or overlaps with an epitope specifically bound by the anti-idiotype antibody or antigen-binding fragment according to any of the embodiments described herein.
In some of any such embodiments, the target antibody or antigen binding fragment is within or contained within an antigen binding domain of the extracellular portion of a Chimeric Antigen Receptor (CAR); and/or the anti-idiotype antibody or antigen-binding fragment specifically binds to a target antibody or antigen-binding fragment contained within or within the antigen-binding domain of the extracellular portion of the CAR. In some embodiments, the target antibody or antigen-binding fragment is an scFv, and the anti-idiotype antibody or antigen-binding fragment specifically binds to an epitope in the scFv of the CAR.
In some of any such embodiments, the antibody or fragment specifically binds to a single chain variable fragment (scFv) derived from antibody SJ25C1 comprised in the extracellular portion of the chimeric antigen receptor, optionally wherein the scFv derived from antibody SJ25C1 comprises the heavy chain variable region set forth in SEQ ID No. 23 and/or the light chain variable region set forth in SEQ ID No. 24; and/or contains the amino acid sequence set forth in SEQ ID NO. 28. In some of any such embodiments, the antibody or fragment specifically binds to a single chain variable fragment (scFv) derived from antibody FMC63 comprised in the extracellular portion of the chimeric antigen receptor, optionally wherein the scFv derived from antibody FMC63 comprises the heavy chain variable region set forth in SEQ ID No. 30 and/or the light chain variable region set forth in SEQ ID No. 31; and/or contains the amino acid sequence set forth in SEQ ID NO. 34.
In some of any such embodiments, the anti-idiotype antibody or antigen-binding fragment specifically binds to an epitope within or including all or part of the Complementarity Determining Regions (CDRs) of the target antibody or antigen-binding fragment.
In some of any such embodiments, the CAR further comprises a transmembrane domain linked to the antigen binding domain by a spacer. In some embodiments, the spacer contains an extracellular portion from CD28, optionally the CD28 is human CD28. In some aspects, the extracellular portion from CD28 contains the amino acid sequence set forth in SEQ ID NO. 27. In some of any such embodiments, the transmembrane domain comprises a transmembrane portion of CD28, which CD28 is optionally human CD28. In some of any such embodiments, the antibody or fragment does not bind to an epitope in the spacer domain of the CAR.
In some of any such embodiments, the antibody or fragment does not bind or does not specifically bind to CD28 or a portion thereof, said CD28 optionally being human CD28, optionally comprising an extracellular portion of CD28, optionally comprising the amino acid sequence set forth in SEQ ID No. 27. In some of any such embodiments, the antibody or fragment does not bind to an epitope in an Fc domain, optionally a human IgG1Fc domain.
In some of any such embodiments, the target antibody or antigen binding fragment specifically binds to human CD 19. In some of any such embodiments, the anti-idiotype antibody or fragment does not cross-react with another anti-CD 19 antibody, which anti-CD 19 antibody is optionally contained in the extracellular antigen-binding domain of another CAR. In some of any such embodiments, the anti-idiotype antibody or fragment does not cross-react with another CAR.
In some of any such embodiments, the anti-idiotype antibody or fragment is an agonist antibody of the CAR that contains the target antibody or antigen binding fragment. In some of any such embodiments, the antibody or fragment is an antagonist of a CAR containing the target antibody or antigen binding fragment.
In some of any such embodiments, the anti-idiotype antibody or antigen-binding fragment thereof is humanized. In some of any such embodiments, the anti-idiotype antibody or antigen-binding fragment thereof is recombinant. In some of any such embodiments, the anti-idiotype antibody or antigen-binding fragment thereof is monoclonal.
In some of any such embodiments, the anti-idiotype antibody or antigen-binding fragment thereof is an antigen-binding fragment. In some aspects, the antigen binding fragment is selected from the group consisting of fragment antigen binding (Fab) fragments, F (ab') 2 Fragments, fab' fragments, fv fragments, single chain variable fragments (scFv), or single domain antibodies.
In some of any such embodiments, the anti-idiotype antibody or antigen-binding fragment thereof comprises at least a portion of an immunoglobulin constant region. In some embodiments, at least a portion of the immunoglobulin constant region comprises a portion of an Fc region or an Fc comprising CH2 and CH3 domains. In some aspects, the constant region is derived from human IgG. In some of any such embodiments, the anti-idiotype antibody or antigen-binding fragment is an intact antibody or a full-length antibody.
In some of any such embodiments, conjugates are provided comprising an anti-idiotype antibody or antigen-binding fragment according to any of the embodiments above and a heterologous molecule or moiety. In some embodiments, the heterologous molecule or module is a label. In some aspects, the label is selected from the group consisting of a fluorescent dye, a fluorescent protein, a radioisotope, a chromophore, a metal ion, a gold particle, a silver particle, a magnetic particle, a polypeptide, an enzyme, streptavidin, biotin, a luminescent compound, or an oligonucleotide. In some cases, the heterologous molecule or module is a protein, peptide, nucleic acid, or small molecule, which is optionally or contains a toxin Strep-Tag.
In some embodiments, nucleic acid molecules encoding the heavy and/or light chains of an anti-idiotype antibody or antigen-binding fragment thereof according to any of the embodiments described herein are provided. In some aspects, the nucleic acid molecule comprises a nucleotide sequence set forth in SEQ ID NO. 15 encoding (i) a heavy chain variable region, (ii) a nucleotide sequence having at least 90% sequence identity to the nucleotide sequence set forth in SEQ ID NO. 15; or (iii) a degenerate sequence of (i) or (ii); and/or the nucleotide sequence set forth in SEQ ID NO. 19 encoding (iv) a light chain variable region, (v) a nucleotide sequence having at least 90% sequence identity to the nucleotide sequence set forth in SEQ ID NO. 19; or (vi) a degenerate sequence of (iv) or (v).
In some of any of these embodiments, the nucleic acid molecule comprises the nucleotide sequence set forth in SEQ ID NO. 17 encoding (i) a heavy chain, (ii) a nucleotide sequence having at least 90% sequence identity to the nucleotide sequence set forth in SEQ ID NO. 17; or (iii) a degenerate sequence of (i) or (ii); and/or the nucleotide sequence set forth in SEQ ID NO. 21 encoding (iv) a light chain, (v) a nucleotide sequence having at least 90% sequence identity to the nucleotide sequence set forth in SEQ ID NO. 21; or (vi) a degenerate sequence of (iv) or (v).
In some embodiments, the nucleic acid molecule comprises a nucleotide sequence set forth in SEQ ID NO. 50 or 71 encoding (i) a heavy chain variable region, (ii) a nucleotide sequence having at least 90% sequence identity to the nucleotide sequence set forth in SEQ ID NO. 50 or 71; or (iii) a degenerate sequence of (i) or (ii); and/or the nucleotide sequence set forth in SEQ ID NO. 54 or 75 encoding (iv) a light chain variable region, (v) a nucleotide sequence having at least 90% sequence identity to the nucleotide sequence set forth in SEQ ID NO. 54 or 75; or (vi) a degenerate sequence of (iv) or (v). In some embodiments, the nucleic acid molecule comprises a nucleotide sequence set forth in SEQ ID NO. 52 or 73 encoding (i) a heavy chain, (ii) a nucleotide sequence having at least 90% sequence identity to the nucleotide sequence set forth in SEQ ID NO. 52 or 73; or (iii) a degenerate sequence of (i) or (ii); and/or the nucleotide sequence set forth in SEQ ID NO. 56 or 76 encoding (iv) a light chain, (v) a nucleotide sequence having at least 90% sequence identity to the nucleotide sequence set forth in SEQ ID NO. 56 or 76; or (vi) a degenerate sequence of (iv) or (v). In some embodiments, the nucleotide sequence encoding the heavy chain and/or the light chain comprises a signal sequence.
Provided herein are vectors containing a nucleic acid molecule according to any of the embodiments described herein. Also provided herein are cells containing an anti-idiotype antibody or antigen-binding fragment thereof according to any of the embodiments described herein or a nucleic acid molecule according to any of the embodiments described herein.
Provided herein are methods of producing an anti-idiotype antibody or antigen-binding fragment thereof, comprising expressing in a suitable host cell a heavy chain and/or a light chain encoded by a nucleic acid molecule according to any of the embodiments described herein or a vector according to any of the embodiments described herein, and recovering or isolating the antibody. In some embodiments, the method of producing an anti-idiotype antibody or antigen-binding fragment comprises culturing a cell according to any of the embodiments described herein under conditions that express the heavy and/or light chain, and recovering or isolating the antibody. Also provided herein are anti-idiotype antibodies or antigen-binding fragments thereof produced by a method according to any of the embodiments described herein.
In some embodiments, there is provided a composition comprising an anti-idiotype antibody or antigen-binding fragment thereof according to any of the embodiments described herein, a conjugate according to any of the embodiments described herein, or a cell according to any of the embodiments described herein. In some of any such embodiments, the composition further comprises a pharmaceutically acceptable excipient.
In some embodiments, kits are provided that contain an anti-idiotype antibody or antigen-binding fragment thereof according to any of the embodiments described herein, a conjugate according to any of the embodiments described herein, one or more of the nucleic acids according to any of the embodiments described herein, and optionally instructions for use. In some cases, the kit further comprises a reagent or support for immobilizing the anti-idiotype antibody or antigen-binding fragment or conjugate thereof, wherein the reagent or support is a bead, column, microwell, stick, filter, strip, or soluble oligomeric streptavidin mutein reagent.
Also provided are detection methods using any of the provided agents (e.g., anti-idiotype antibodies). In some embodiments, methods of detecting a target antibody or antigen-binding fragment thereof (e.g., a CAR containing it) are provided, the methods comprising (a) contacting a composition comprising a target antibody (e.g., an antibody having a variable region derived from antibody SJ25C1 or from antibody FMC63, or an antigen-binding fragment from one of such antibodies) with an anti-idiotype antibody or antigen-binding fragment thereof; (b) Detecting an anti-idiotype antibody bound to a target antibody or antigen-binding fragment and/or detecting the presence or absence of a target antibody or agent.
In some embodiments, the method comprises (a) contacting a composition comprising or suspected of comprising a target antibody (which is antibody FMC63 or an antigen binding fragment thereof) with the anti-idiotype antibody of any of the embodiments or an antigen binding fragment thereof or the conjugate of any of the embodiments, which specifically binds to the target antibody (which is antibody FMC63 or an antigen binding fragment thereof); (b) Detecting an anti-idiotype antibody bound to a target antibody or antigen-binding fragment and/or detecting the presence or absence of a target antibody or agent. .
In some aspects, the target antibody or antigen binding fragment binds to or is expressed on the surface of a cell, and the detecting in (b) comprises detecting a cell that binds to the anti-idiotype antibody. In some cases, the cell expresses a CAR containing the target antibody or target antigen binding fragment on its surface.
In some embodiments, provided methods comprise detecting a CAR comprising the target antibody or antigen binding fragment thereof of any of the embodiments, such as a CAR comprising a variable domain derived from FMC63 or SJ 25C. In some aspects, the method comprises (a) contacting a cell expressing a Chimeric Antigen Receptor (CAR) containing a target antibody (which is antibody SJ25C1 or antigen binding fragment thereof) with an anti-idiotype antibody according to any of the embodiments or antigen binding fragment thereof or a conjugate according to any of the embodiments, which specifically binds to the target antibody (which is antibody SJ25C1 or antigen binding fragment thereof); and (b) detecting cells that bind to the anti-idiotype antibody. In some embodiments, the method comprises (a) contacting a cell expressing a Chimeric Antigen Receptor (CAR) containing a target antibody (which is antibody FMC63 or antigen binding fragment thereof) with the anti-idiotype antibody of any of the embodiments or antigen binding fragment thereof or the conjugate of any of the embodiments, which specifically binds to the target antibody (which is antibody FMC63 or antigen binding fragment thereof); and (b) detecting cells that bind to the anti-idiotype antibody. In some of any such embodiments, the anti-idiotype antibody or antigen-binding fragment thereof is labeled, directly or indirectly, for detection.
In some embodiments, there is provided a method of selecting cells from a population of cells, the method comprising (a) contacting a population of cells expressing a Chimeric Antigen Receptor (CAR) containing a target antibody, or cells that bind to a target antibody, with an anti-idiotype antibody according to any of the embodiments described herein, or an antigen-binding fragment thereof, or a conjugate according to any of the embodiments, that specifically binds to a target antibody (which is antibody SJ25C1, or an antigen-binding fragment thereof), wherein the target antibody is antibody SJ25C1, or an antigen-binding fragment thereof; and (b) selecting cells that bind to the anti-idiotype antibody. In some embodiments, the method comprises (a) contacting a population of cells expressing a Chimeric Antigen Receptor (CAR) comprising a target antibody, or cells that bind to a target antibody, with an anti-idiotype antibody of any of the embodiments, or an antigen-binding fragment thereof, or a conjugate of any of the embodiments, that specifically binds to a target antibody (which is antibody FMC63, or an antigen-binding fragment thereof), wherein the target antibody is antibody FMC63, or an antigen-binding fragment thereof; and (b) selecting cells that bind to the anti-idiotype antibody.
In some cases, cells that bind to the anti-idiotype antibody are selected by affinity-based separation. In some aspects, the affinity-based separation is an immunoaffinity-based separation. In some of any such embodiments, the affinity-based separation is accomplished by flow cytometry. In some embodiments, affinity-based separation is accomplished by magnetically activated cell sorting. In some aspects, affinity-based separation comprises affinity chromatography. In some of any such embodiments, the anti-idiotype antibody is reversibly bound or immobilized to a support or stationary phase.
Among the methods provided are also methods of stimulating cells using the agents, such as stimulating cells containing molecules such as CARs that are or contain target antibodies recognized by anti-idiotype antibodies. In some aspects, the methods involve incubating an input composition of cells expressing a Chimeric Antigen Receptor (CAR) containing a target antibody (which is antibody SJ25C1 or antigen binding fragment thereof) with an anti-idiotype antibody according to any of the embodiments or antigen binding fragment thereof or conjugate of any of the embodiments that specifically binds to the target antibody (which is antibody SJ25C1 or antigen binding fragment thereof), thereby generating an output composition containing stimulated cells. In some embodiments, the method comprises incubating an input composition of cells expressing a Chimeric Antigen Receptor (CAR) containing a target antibody (which is antibody FMC63 or antigen binding fragment thereof) with the anti-idiotype antibody of any of the embodiments or antigen binding fragment thereof or conjugate of any of the embodiments that specifically binds to the target antibody (which is antibody FMC63 or antigen binding fragment thereof), thereby generating an output composition containing stimulated cells.
In some embodiments, the methods result in proliferation, activation, stimulation, cytokine release, or other functional outcome, such as upregulation of an activation marker or cytokine release or production, of cells expressing a chimeric receptor (such as a CAR recognized by an anti-idiotype antibody). In some aspects, the extent to which such proliferation or other functional response or readout is induced in such cells is similar to or greater than that induced by incubating the cells with agents and/or conditions that stimulate T cell proliferation (e.g., anti-CD 3/CD28 beads and/or cross-linked anti-CD 3). In some aspects, the methods do not involve cross-linking of anti-idiotype antibodies. In some aspects of any embodiment, the anti-idiotype agent is capable of inducing a particular proliferation or functional outcome or degree thereof without cross-linking the anti-idiotype antibody. In some aspects, the anti-idiotype agents herein are advantageous in terms of their ability to stimulate or elicit specific functional consequences for T cells or other immune cells expressing the target receptor without the need to crosslink the anti-Id antibody or use a second agent. In some aspects, the results are achieved with anti-idiotype antibodies in soluble or plate-bound form. In some aspects, the results are achieved with anti-idiotype antibodies conjugated to beads.
In some embodiments, methods of producing a cellular composition are provided, the methods comprising (a) introducing a nucleic acid molecule encoding a Chimeric Antigen Receptor (CAR) into a cell, thereby producing an input composition; and (b) incubating the input composition with an anti-idiotype antibody or antigen-binding fragment thereof specific for an antigen receptor CAR, thereby producing a cellular composition.
In some aspects, the CAR contains a target antibody that specifically binds to CD 19. In some embodiments, the target antibody is antibody SJ25C1 or an antigen binding fragment thereof. In some of any such embodiments, the anti-idiotype antibody or antigen-binding fragment thereof is an anti-idiotype antibody or antigen-binding fragment thereof according to any of the embodiments, which specifically binds to a target antibody (which is antibody SJ25C1 or antigen-binding fragment thereof). In some cases, the target antibody is antibody FMC63 or an antigen binding fragment thereof. In some of any such embodiments, the anti-idiotype antibody or antigen-binding fragment thereof specifically binds to the target antibody (which is antibody FMC63 of any of the embodiments), and the anti-idiotype antibody or antigen-binding fragment thereof specifically binds to the target antibody (which is antibody FMC63 or antigen-binding fragment thereof).
In some of any such embodiments, the introducing in (a) comprises introducing the nucleic acid molecule into the cell by viral transduction, transposition, electroporation, or chemical transfection. In some cases, the introducing in (a) comprises introducing the nucleic acid molecule into the cell by transduction with a retroviral vector comprising the nucleic acid molecule, optionally wherein the viral vector is a retroviral vector or a lentiviral vector. In some aspects, the introducing in (a) comprises introducing the nucleic acid molecule into the cell by transposing with a transposon containing the nucleic acid molecule. In some cases, the introducing in (a) comprises introducing the nucleic acid molecule into the cell by electroporation or transfection of a vector comprising the nucleic acid molecule.
In some of any such embodiments, the method further comprises the step of activating the cells prior to step (a). In some aspects, the step of activating the cell comprises contacting the cell with a CD3 agonist and optionally a CD28 agonist. In some cases, the step of activating the cells comprises contacting the cells with an agent comprising an agonist anti-CD 3 antibody and an anti-CD 28 antibody.
In some of any such embodiments, the incubation is performed under conditions in which the anti-idiotype antibody or antigen-binding fragment thereof binds to the CAR, thereby inducing or modulating a signal in one or more cells of the input composition. In any such embodiment, the cell comprises a T cell. In some cases, the T cells contain cd4+ and/or cd8+ T cells.
In some of any such embodiments, the anti-idiotype antibody or antigen-binding fragment thereof is immobilized to a solid support, optionally containing or conjugated to an agent containing a plurality of binding sites capable of reversibly binding to the anti-idiotype antibody or antigen-binding fragment thereof. In some of any such embodiments, the anti-idiotype antibody or antigen-binding fragment thereof is immobilized to a soluble agent, optionally or containing a plurality of binding sites capable of reversibly binding to the anti-idiotype antibody or antigen-binding fragment thereof. In some aspects, the reagent contains a streptavidin mutein.
In some of any such embodiments, the incubation is at least or about at least 5 minutes, 10 minutes, 30 minutes, 60 minutes, 2 hours, 6 hours, 12 hours, 24 hours, 36 hours, 48 hours, 72 hours, or 96 hours.
In some of any such embodiments, the input composition contains less than or less than about 60%, less than or less than about 50%, less than or less than about 40%, less than or less than about 30%, less than or less than about 20%, or less than about 10% of CAR-expressing cells as a percentage of the total cells in the composition.
In some of any such embodiments, the number of cells expressing the CAR in the output composition is increased by greater than 1.2-fold, 1.5-fold, 2.0-fold, 3.0-fold, 4.0-fold, 5.0-fold, 10-fold, or more as compared to the number of cells expressing the CAR in the input composition; and/or the percentage of CAR-expressing cells in the output composition is increased by greater than 10%, 20%, 40%, 50%, 60%, 70%, 80% or more as compared to the total cells in the composition.
In some of any such embodiments, the CAR-expressing cells are not selected or enriched for the cells prior to introduction and/or incubation.
In some of any such embodiments, the target antibody or antigen-binding fragment contains the heavy chain variable region set forth in SEQ ID NO. 23 and/or the light chain variable region set forth in SEQ ID NO. 24. In some of any such embodiments, the target antibody or antigen-binding fragment contains the heavy chain variable region set forth in SEQ ID NO. 30 and/or the light chain variable region set forth in SEQ ID NO. 31.
In some embodiments, there is provided a method of purifying an antibody or antigen-binding fragment thereof, the method comprising (a) contacting a composition comprising a target antibody (which is antibody SJ25C1 or antigen-binding fragment thereof) with an anti-idiotype antibody or antigen-binding fragment thereof according to any of the embodiments described herein or a conjugate according to any of the embodiments, which specifically binds to the target antibody (which is antibody SJ25C1 or antigen-binding fragment thereof); and (b) isolating the complex containing the anti-idiotype antibody. In some embodiments, the method comprises (a) contacting a composition comprising a target antibody (which is antibody FMC63 or an antigen binding fragment thereof) with the anti-idiotype antibody of any of the embodiments or an antigen binding fragment thereof or the conjugate of any of the embodiments, which specifically binds to the target antibody (which is antibody FMC63 or an antigen binding fragment thereof); and (b) isolating the complex comprising the anti-idiotype antibody.
In some embodiments, complexes containing anti-idiotype antibodies are separated by affinity-based separation. In some aspects, the affinity-based separation is an immunoaffinity-based separation. In some cases, the affinity-based separation is a magnetic-based separation. In some embodiments, the affinity-based separation comprises affinity chromatography.
In some embodiments, methods of identifying an anti-idiotype antibody or antigen-binding fragment are provided, the methods comprising (a) introducing into a subject a soluble immunoreagent comprising an antigen-binding fragment of a target antibody fused to a solubilizing moiety; and (b) identifying antibodies from the subject that specifically bind to the target antibody or antigen-binding fragment thereof.
In some of any such embodiments, the antigen binding fragment comprises a variable heavy chain region and/or a variable light chain region of the target antibody. In some embodiments, the antigen binding fragment is a single-stranded fragment. In some aspects, the antigen binding fragment is an scFv. In some of any such embodiments, the antigen binding fragment is within or contained within an antigen binding domain of the extracellular portion of a Chimeric Antigen Receptor (CAR).
In some of any such embodiments, the solubilising moiety is an Fc domain or fragment thereof, optionally a human IgG1Fc. In some aspects, the solubilizing module is an Fc domain lacking a hinge region. In some cases, the solubilizing module contains the amino acid sequence set forth in SEQ ID NO. 32.
In some of any such embodiments, identifying the antibody comprises (i) isolating B cells from the spleen of the subject and fusing them with immortalized B cells to generate hybridomas; (ii) Screening the hybridomas for the production of antibodies that specifically bind to the target antibody or antigen-binding fragment thereof or chimeric antigen receptor comprising the antigen-binding fragment; and (iii) sequencing the antibodies from the hybridomas producing the specifically bound antibodies, thereby identifying the anti-idiotype antibodies.
In some of any such embodiments, the target antibody binds to CD 19. In some embodiments, the antigen-binding fragment of the target antibody is derived from antibody SJ25C1, optionally wherein the antigen-binding fragment of the target antibody comprises the heavy chain variable region set forth in SEQ ID No. 23 and/or the light chain variable region set forth in SEQ ID No. 24. In some of any such embodiments, the antigen-binding fragment of the target antibody is a single chain variable fragment (scFv) derived from antibody SJ25C1, optionally wherein the scFv comprises the amino acid sequence set forth in SEQ ID No. 28. In some embodiments, the antigen-binding fragment of the target antibody is derived from antibody FMC63, optionally wherein the antigen-binding fragment of the target antibody comprises the heavy chain variable region set forth in SEQ ID No. 30 and/or the light chain variable region set forth in SEQ ID No. 31. In some embodiments, the antigen-binding fragment of the target antibody is a single chain variable fragment (scFv) derived from the antibody FMC63, optionally wherein the scFv comprises the amino acid sequence set forth in SEQ ID NO. 34.
In some embodiments, there is provided a method of depleting cells, the method comprising administering to a subject a composition comprising an anti-idiotype antibody according to any of the embodiments described herein, or an antigen-binding fragment thereof, or a conjugate according to any of the embodiments, that specifically binds to a target antibody (which is antibody SJ25C1 or an antigen-binding fragment thereof), wherein the subject has been administered cells expressing a Chimeric Antigen Receptor (CAR) comprising the target antibody, which is antibody SJ25C1 or an antigen-binding fragment thereof. In some embodiments, the method comprises administering to a subject a composition comprising an anti-idiotype antibody of any of the embodiments described herein, or an antigen-binding fragment thereof, or a conjugate of any of the embodiments, that specifically binds to a target antibody (which is antibody FMC63 or an antigen-binding fragment thereof), wherein the subject has been administered a cell that expresses a Chimeric Antigen Receptor (CAR) containing the target antibody, which is antibody FMC63 or an antigen-binding fragment thereof. In some embodiments, depletion occurs by antibody-dependent cell-mediated cytotoxicity (ADCC).
Provided herein are methods of determining the presence or absence of a molecule that binds to a Chimeric Antigen Receptor (CAR), the method comprising (a) contacting a binding agent with a sample from a subject to which a cell therapy has been administered under conditions that form a complex comprising the binding agent and a molecule from the sample that binds to the binding agent, the cell therapy comprising a cell engineered with a CAR comprising a target antibody that is antibody SJ25C1 or an antigen binding fragment thereof, wherein the binding agent comprises an extracellular domain of a CAR comprising the target antibody or an antigen binding fragment thereof, or a portion thereof; and (b) detecting the presence or absence of the complex, thereby determining the presence or absence of a molecule that binds to the CAR. In some embodiments, the method further comprises performing steps (a) and (b) on a positive control sample, and optionally determining the presence or absence of the molecule by comparison to a positive control sample, wherein the positive control sample contains any anti-idiotype antibody or antigen-binding fragment thereof described herein or any conjugate described herein that specifically binds to a target antibody or antigen-binding fragment thereof.
Provided herein are methods of determining the presence or absence of a molecule that binds to a Chimeric Antigen Receptor (CAR), the method comprising (a) contacting a binding agent with a sample from a subject to which a cell therapy has been administered under conditions that form a complex comprising the binding agent and a molecule from the sample that binds to the binding agent, the cell therapy comprising a cell engineered with a CAR comprising a target antibody that is antibody FMC63 or an antigen binding fragment thereof, wherein the binding agent comprises an extracellular domain of a CAR comprising the target antibody or an antigen binding fragment thereof, or a portion thereof; and (b) detecting the presence or absence of the complex. In some embodiments, the method further comprises performing steps (a) and (b) on a positive control sample, and optionally determining the presence or absence of the molecule by comparison to a positive control sample, wherein the positive control sample contains any anti-idiotype antibody or antigen-binding fragment thereof or any conjugate described herein that specifically binds to a target antibody or antigen-binding fragment thereof.
In some of any such embodiments, the molecule that binds to the binding agent is or comprises an antibody. In some embodiments, the binding reagent is subjected to a detectable label or is capable of producing a detectable signal. In some cases, the binding reagent is bound to or soluble in the solid support.
In some of any such embodiments, the complex is detected by an immunoassay. In some examples, the immunoassay is an enzyme-linked immunosorbent assay (ELISA), a chemiluminescent assay, an electrochemiluminescent assay, a Surface Plasmon Resonance (SPR) based biosensor (e.g., BIAcore), flow cytometry, or Western blot. In some embodiments, the immunoassay comprises mesoscale discovery. In some cases, the immunoassay is a sandwich assay or a bridging assay.
In some of any such embodiments, the binding reagent is a first binding reagent, and detecting the presence or absence of a complex comprises: (i) Contacting the complex formed in step (a) with a second binding agent, wherein the second binding agent (1) comprises an extracellular domain of a CAR comprising a target antibody or antigen-binding fragment thereof, or a portion thereof, and (2) is detectably labeled or capable of producing a detectable signal; and (ii) assessing the presence or absence of the detectable signal. In some aspects, the first binding reagent is bound to a solid support, optionally wherein the first binding reagent is directly or indirectly linked to biotin and/or bound to a solid support via streptavidin; and/or the second binding reagent is soluble. In some cases, the extracellular domains of the CARs of the first and second binding agents, or portions thereof, are identical.
In some of any such embodiments, the detectable label is or comprises a fluorescent label, a chemiluminescent label, an electroluminescent label, a colorimetric label, a bioluminescent label, or a radioactive label; and/or the detectable signal is or comprises a fluorescent signal, a chemiluminescent signal, an electroluminescent signal, a colorimetric signal, a bioluminescent signal, or a radioactive signal. In some of any such embodiments, the detectable label is or comprises a sulphur-TAG.
In some of any such embodiments, the antigen-binding fragment of the target antibody comprises a variable heavy chain region and/or a variable light chain region of the target antibody. In some of any such embodiments, the antigen-binding fragment of the target antibody is a single-chain fragment. In some embodiments, the antigen-binding fragment of the target antibody is an scFv.
In some of any such embodiments, the sample comprises whole blood, serum, or plasma.
Provided herein are articles of manufacture comprising any of the anti-idiotype antibodies described herein, or antigen-binding fragments thereof, or any of the conjugates, for detecting SJ25C1 antibodies, or antigen-binding fragments thereof, or chimeric antigen receptors comprising SJ25C1 antibodies, or antigen-binding fragments thereof, and instructions for using the anti-idiotype antibodies; selecting or enriching from a population of cells an engineered cell expressing a Chimeric Antigen Receptor (CAR) comprising antibody SJ25C1 or an antigen binding fragment thereof; an input composition comprising cells expressing a chimeric antigen receptor comprising an SJ25C1 antibody or antigen binding fragment thereof is stimulated.
Provided herein are articles of manufacture comprising any anti-idiotype antibody or antigen-binding fragment thereof or any conjugate described herein as described herein, and instructions for using the anti-idiotype antibody for detecting FMC63 antibodies or antigen-binding fragments thereof or chimeric antigen receptors comprising FMC63 antibodies or antigen-binding fragments thereof; selecting or enriching from a population of cells an engineered cell expressing a Chimeric Antigen Receptor (CAR) comprising the antibody FMC63 or an antigen binding fragment thereof; an input composition comprising cells expressing a chimeric antigen receptor comprising an FMC63 antibody or antigen binding fragment thereof is stimulated.
Provided herein are articles of manufacture comprising a binding agent comprising an extracellular domain of a Chimeric Antigen Receptor (CAR) comprising a target antibody (which is antibody FMC63 or antigen binding fragment thereof), the extracellular domain or portion thereof comprising the target antibody or antigen binding fragment thereof; and an anti-idiotype antibody or antigen-binding fragment described herein or any conjugate described herein. In some embodiments, the binding agent is a first binding agent, and the article further comprises a second binding agent comprising an extracellular domain of a CAR or a portion thereof.
In some of any such embodiments, the extracellular domains of the CARs of the first and second binding agents, or portions thereof, are identical.
In some of any such embodiments, the article of manufacture further comprises instructions for using the binding reagent (optionally the first and second binding reagents) to determine the presence or absence of a molecule that binds to the binding reagent in a sample by using an immunoassay, optionally wherein the immunoassay is a bridging or sandwich immunoassay, optionally wherein the sample is from a subject who has been administered a cell therapy comprising a cell engineered with a CAR comprising a target antibody that is antibody FMC63 or an antigen binding fragment thereof.
Provided herein are articles of manufacture comprising a binding agent comprising an extracellular domain of a Chimeric Antigen Receptor (CAR) comprising a target antibody (which is antibody SJ25C1 or antigen binding fragment thereof), the extracellular domain or portion thereof comprising a target antibody or antigen binding fragment thereof; and an anti-idiotype antibody or antigen-binding fragment described herein or a conjugate described herein.
In some embodiments, the binding agent is a first binding agent, and the article of manufacture further comprises a second binding agent comprising an extracellular domain of a CAR or a portion thereof. In some aspects, the extracellular domains of the CARs of the first and second binding agents, or portions thereof, are identical.
In some of any such embodiments, the article of manufacture further comprises instructions for using the binding reagent (optionally the first and second binding reagents) to determine the presence or absence of a molecule bound to the binding reagent in a sample by using an immunoassay, optionally wherein the immunoassay is a bridging or sandwich immunoassay, optionally wherein the sample is from a subject who has been administered a cell therapy comprising a cell engineered with a CAR comprising a target antibody, which is antibody SJ25C1 or antigen binding fragment thereof.
In some embodiments, the binding reagent (optionally the first and/or second binding reagent) is labeled or capable of producing a detectable signal. In some cases, one of the first and second binding reagents is or is capable of being attached to a solid support and the other of the first and second binding reagents is or is capable of producing a detectable signal. In some embodiments, the article further comprises a solid support, optionally wherein one of the first and second binding reagents is directly or indirectly attached to biotin, and the solid support comprises a streptavidin-coated surface.
Brief Description of Drawings
FIG. 1 shows the results of flow cytometry for evaluating the functional activity of SJ25C 1-derived scFv-specific anti-idiotype antibody clone A-1 (anti-idiotype antibody A-1) to stimulate Erk1/2 phosphorylation in Jurkat cells engineered with SJ25C 1-derived CARs. Activation with anti-CD 3 antibody was included as a positive control. Non-stimulated cells or isotype control stimulated cells were included as negative controls.
FIGS. 2A-B show proliferation assay results of T cells expressing CARs containing either SJ25C 1-derived binding domain (FIG. 2A) or FMC 63-derived binding domain (FIG. 2B), as assessed by dye dilution using flow cytometry after stimulation with anti-CD 3 antibody (OKT 3), anti-ID A-1 or anti-ID B-1 anti-idiotype antibody. Unstimulated cells were included as negative controls.
Figure 2C shows the results of an assay for simulated transduction and proliferation of CAR transduction after T cells (labeled with dye) are cultured in the presence of stimulation by a plate-bound anti-idiotype antibody that recognizes the binding domain of the CAR, as assessed by dye dilution using flow cytometry.
FIG. 3 shows the results of an assessment of the expression of two T cell activation markers CD69 and CD25 in CD4+ or CD8+ T cells expressing a CAR having a variable region derived from SJ25C1 as assessed by flow cytometry after stimulation with a plate-bound anti-CD 3 antibody (OKT 3), anti-ID A-1 or anti-ID B-1.
FIG. 4 shows the results of an assessment of the expression of two T cell activation markers CD69 and CD25 in CD4+ or CD8+ T cells expressing a CAR having a binding domain (with variable region derived from FMC 63) as assessed by flow cytometry after stimulation with a plate-bound anti-CD 3 antibody (OKT 3), anti-ID A-1 or anti-ID B-1 or negative control non-target anti-idiotype antibody. Unstimulated cells were included as negative controls.
FIG. 5 shows the results of a bridge ELISA for detection of anti-CAR antibodies using anti-ID B-1 and anti-ID B-2 antibodies recognizing the CAR binding domain over a range of concentrations as positive controls.
FIGS. 6 and 7 show the expansion and accumulation cell numbers of EGFRt+/CD4+ T cells or EGFRt+/CD8+ T cells stimulated with a indicated proportion of anti-idiotype antibody (anti-ID B-1) coated beads or control CD3/CD28 antibody coated beads, respectively, in the presence or absence of cytokines.
FIG. 8 shows the PD-1 expression levels of CD4+ T cells positive for anti-EGFR antibody staining and thus positive for the transduction marker EGFR in the presence or absence of cytokines after stimulation with either anti-idiotype antibody (anti-ID B-1) coated beads or control CD3/CD28 antibody coated beads at the indicated ratio, as assessed by flow cytometry on days 3, 7, 10 and 14 of culture.
Figure 9 shows CD4 expressing FMC 63-derived CAR as assessed by flow cytometry + Or the viability of cd8+ T cells after stimulation with a specified proportion of anti-idiotype antibody (anti-ID B-1) coated beads or control CD3/CD28 antibody coated beads in the presence or absence of cytokines, as assessed by flow cytometry on days 3, 7, 10 and 14 of culture.
FIG. 10A shows intracellular cytokine staining for IL-2, TNF alpha and IFN gamma following stimulation with FMC 63-derived scFv-specific anti-idiotype antibody (anti-ID B-1) coated beads of T cells expressing FMC 63-derived CARs. Results for cd8+ T cells positive or negative for EGFRt surrogate transduction markers (egfr+ or EGFRt-) are shown.
Figure 10B shows intracellular cytokine staining for IL-2, tnfα and ifnγ following stimulation with K562-CD19 cells expressing antigen for T cells expressing FMC 63-derived CARs. Results are shown for cd8+ T cells positive for anti-EGFR antibodies as a surrogate for CAR expression.
FIG. 11 shows population doublings in a continuous stimulation assay of T cells expressing FMC 63-derived scFv-derived CAR in the presence or absence of cytokines following stimulation with a specified proportion of anti-idiotype antibody (anti-ID B-1) coated beads or control anti-CD 3/anti-CD 28 antibody coated beads over a 14 day culture period. Results are shown for cd4+ T cells positive for EGFRt instead of transduction markers (egfrt+/cd4+) or for cd8+ T cells positive for EGFRt (egfrt+/cd8+).
FIGS. 12A-12C show the results after stimulation of CD4+ or CD8+ T cells expressing FMC 63-derived CARs, either alone or as co-cultures with FMC 63-derived scFv-specific anti-idiotype antibody (anti-ID B-1) coated beads. Results for two different donors are shown. FIG. 12A depicts the fold expansion of CD4+ T cells or CD8+ T cells (EGFRt+/CD4+ or EGFRt+/CD8+) positive for EGFRt-alternative transduction markers in culture. FIG. 12B shows the frequency of EGFRt positive CD4+ T cells or CD8+ T cells (EGFRt+/CD4+ or EGFRt+/CD8+) in culture. Figure 12C shows the viability of cd4+ T cells or cd8+ T cells in culture.
Figures 13A and 13B show the results of flow cytometry on T cell surface markers on days 5, 7 and 9 of culture following stimulation of cd4+ or cd8+ T cells expressing FMC 63-derived CARs, either alone or as co-cultures with FMC 63-derived scFv-specific anti-idiotype antibody (anti-ID B-1) coated beads. FIG. 13A shows PD-1 surface expression on CD4+ T cells or CD8+ T cells (EGFRt+/CD4+ or EGFRt+/CD8+) positive for EGFRt alternative transduction markers in culture. Figure 13B shows CD25 surface expression on cd4+ T cells or cd8+ T cells (egfrt+/cd4+ or egfrt+/cd8+) positive for anti-EGFR antibodies as CAR expression substitutes in culture.
Fig. 14A shows intracellular cytokine levels of tnfα, ifnγ and IL-2 of cd4+ or cd8+ T cells assessed by flow cytometry, the cd4+ or cd8+ T cells being present in a thawed composition containing T cells expressing FMC 63-derived CARs that have been expanded in culture with K562 cells expressing CD19 or with PMA/ionomycin. Cytokine levels in cd4+ and cd8+ T cells alone or as co-cultures upon thawing (d=0) or after further 9 days in the presence of anti-ID B-1 conjugated beads are shown.
Figure 14B shows the frequency of cd4+ or cd8+ T cells positive for CD25 or Ki67 assessed by flow cytometry, the cd4+ or cd8+ T cells being present in a thawed composition containing T cells expressing FMC 63-derived CAR that have been expanded in culture with K562 cells expressing CD19 or with PMA/ionomycin. Marker levels in cd4+ and cd8+ T cells alone or as co-cultures were shown upon thawing (d=0) or after further 9 days of culture in the presence of anti-ID B-1 conjugated beads.
FIGS. 15A and 15B show graphs depicting the results of staining cells with anti-EGFR antibodies or FMC 63-derived scFv specific anti-idiotype antibodies (anti-ID B-1 and anti-ID B-2). Fig. 15A shows a graph depicting the average fluorescence intensity of cells stained with different concentrations of antibodies. Cells include a mixture of PBMCs and CAR-expressing cells. Figure 15B shows a graph depicting the percentage of CAR-expressing cells comprising FMC 63-derived scFv specific anti-idiotype antibody (anti-ID B-1) detected in cells stained with different concentrations of antibody. Cells include a mixture of PBMCs and CAR-expressing cells and PBMCs alone.
Detailed Description
Provided herein are agents, such as anti-idiotype antibodies and antigen-binding fragments (e.g., single chain fragments, including scFv), that specifically recognize an anti-CD 19 antibody moiety (e.g., an anti-CD 19 antibody moiety present in a recombinant receptor, including a chimeric antigen receptor). Also provided are uses and methods of use thereof, and compositions and articles of manufacture comprising such agents, including methods for specifically identifying, selecting, and/or stimulating and/or activating cells expressing or comprising a target antibody or fragment, such as anti-CD 19CAR T cells. In some embodiments, the provided antibodies can be used to specifically identify and/or select various anti-CD 19 CARs, e.g., CARs that bind to or are expressed on the surface of a cell, and can also be used to specifically activate cells expressing a target CAR, such as CAR T cells. In some embodiments, antibodies specific for an anti-CD 19 antibody designated SJ25C1 or FMC63, or antibody fragments derived therefrom, are provided, including antibodies and CARs comprising variable regions derived from such antibodies, and/or antibodies comprising the idiotypes contained therein.
In some aspects, the provided anti-idiotype antibodies provide advantages over conventional reagents for detecting, identifying, manipulating, and/or affecting and/or engineering cells expressing a CAR (particularly a CAR containing an anti-CD 19 antibody scFv extracellular domain or a CAR containing a putative idiotype). In certain useful methods, detection of the presence or absence or amount of a CAR or CAR-expressing cell in a sample (and/or stimulation or manipulation of a CAR) is performed by assessing the presence or absence or amount of a surrogate molecule, such as a molecule that is contained in a construct encoding a CAR and thus serves as an indirect or surrogate marker of its expression. In certain available methods, universal antibody reagents are used and/or reagents that are non-specific (e.g., compared to other CARs that may have similar or identical domains other than the antigen binding region) to the particular CAR being evaluated are used for detection; for example, such antibodies can include anti-species antibodies that recognize spacers or other domains from the species from which the CAR domain is derived, and/or antibodies that recognize specific components used in the spacer region of the target, as well as other chimeric receptors. In certain useful methods designed to detect the presence or absence of a CAR, detection is performed using an agent that recognizes the CAR constant region. In certain useful methods, the CAR cells are stimulated by using a universal agent, such as an anti-CD 3/CD28 recognition agent. Some methods use recombinant ligands for the CAR (e.g., CD 19-Fc). In some cases, such methods may not be entirely satisfactory and/or have certain limitations. In some cases, CAR ligands (such as CD 19) may not always be fully effective, for example, in a complex flow cytometry set. Improved methods and agents, including those that provide improved sensitivity and/or selectivity, are needed. Embodiments are provided herein that meet this need.
In some embodiments, the anti-idiotype antibodies and antigen-binding fragments provided overcome the following challenges: the low binding affinity associated with the target antibody ligand and the non-specific binding associated with the antibody reagent directed against the target antibody constant region, thereby providing a reagent having high affinity and specificity for its target antibody or antigen binding fragment thereof. In some embodiments, the provided antibodies exhibit higher specificity and binding affinity for their target antibodies or antigen binding fragments (such as anti-CD 19 antibodies designated SJ25C1 or FMC 63) than CD19-Fc and other reagents currently available for detecting or identifying CARs.
Furthermore, in certain embodiments, the anti-idiotype antibodies and antigen-binding fragments may be selected as agonists or antagonists of chimeric receptors, including target antibodies or antigen-binding fragments thereof, thereby allowing selective detection, isolation, ablation, and/or depletion (e.g., by antibody-dependent cell-mediated cytotoxicity (ADCC) killing), and/or stimulation or activation of cells having such chimeric receptors bound or expressed on their surfaces. Provided herein are anti-idiotype antibody agonists that exhibit activity to stimulate (e.g., activate) CARs containing an extracellular binding domain derived from an anti-CD 19 antibody designated SJ25C1 or FMC 63. In some aspects, such antibodies can be used in methods of stimulating and amplifying cells expressing a specific CAR, including in the process of generating and preparing cells expressing a CAR.
Also provided herein are nucleic acids encoding the provided anti-idiotype antibodies and fragments, as well as cells, such as recombinant cells, that express and produce these anti-idiotype antibodies and fragments. Methods of making and using the anti-idiotype antibodies and fragments, and cells expressing or containing the anti-idiotype antibodies and fragments, are also provided.
All publications mentioned in this specification, including patent documents, scientific papers, and databases, are incorporated herein by reference in their entirety for all purposes to the same extent as if each individual publication was specifically and individually indicated to be incorporated by reference. If a definition set forth herein is contrary to or otherwise inconsistent with a definition set forth in the patents, applications, published applications and other publications that are incorporated by reference, the definition set forth herein takes precedence over the definition set forth herein.
The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.
Anti-idiotype antibody
In some aspects, binding molecules are provided, such as anti-idiotype antibodies or antigen binding fragments ("anti-IDs") that specifically recognize a target anti-CD 19 antibody moiety. In some embodiments, the provided antibodies recognize a target anti-CD 19 antibody that is SJ25C1 or an antigen binding fragment thereof or an antibody or antigen binding fragment derived from SJ25C 1. In some embodiments, the provided antibodies recognize a target anti-CD 19 antibody that is FMC63 or an antigen binding fragment thereof or an antibody or antigen binding fragment derived from FMC 63.
SJ25C1 is a mouse monoclonal IgG1 antibody raised against Nalm-1 and-16 cells expressing human CD19 (Ling, N.R. et al (1987) Leucocyte typing III.302). The SJ25C1 antibody comprises the CDRH1, H2 and H3 sequences set forth in SEQ ID NOS: 114-116, respectively, and the CDRL1, L2 and L3 sequences set forth in SEQ ID NOS: 117-119, respectively. The SJ25C1 antibody comprises a heavy chain variable region (V H ) And a light chain variable region (V) comprising the amino acid sequence SEQ ID NO. 24 L )。
In some embodiments, the target antibody is SJ25C1 or an antibody derived from SJ25C 1. In some embodiments, the antibody derived from SJ25C1 isV comprising SJ25C1 H And/or V L An antibody or antigen binding fragment of an idiotype of SJ25C1, a paratope of SJ25C1, or one or more Complementarity Determining Regions (CDRs) of SJ25C 1. In some embodiments, the target antibody (which is SJ25C1 or an antibody derived from SJ25C 1) is an antibody or antigen binding fragment comprising V of SJ25C1 set forth in SEQ ID NO. 23 H Or a variant thereof having at least 90% sequence identity to SEQ ID NO. 23, and/or V of SJ25C1 set forth in SEQ ID NO. 24 L Or a variant thereof having at least 90% sequence identity to SEQ ID NO. 24. In some embodiments, the target antibody (which is SJ25C1 or an antibody derived from SJ25C 1) is an antibody or antigen binding fragment comprising V of SJ25C1 set forth in SEQ ID NO. 23 H Or a variant thereof having at least 90% sequence identity to SEQ ID NO. 23, and V of SJ25C1 set forth in SEQ ID NO. 24 L Or a variant thereof having at least 90% sequence identity to SEQ ID NO. 24. In some embodiments, the antibody or antigen binding fragment comprises V of SJ25C1 set forth in SEQ ID NO. 23 and SEQ ID NO. 24, respectively H And V L . In some embodiments, the variant has at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO. 23 and/or SEQ ID NO. 24.
In some embodiments, the target antibody (which is SJ25C1 or an antibody derived from SJ25C 1) is an antibody or antigen binding fragment comprising SJ25C1V set forth in SEQ ID NO. 23 H Is a heavy chain CDR (CDR-H) (such as set forth in SEQ ID NOS: 114-116), and/or SJ25C1V set forth in SEQ ID NO: 24) L Is a light chain CDR (CDR-L) (such as set forth in SEQ ID NOS: 117-119). In some embodiments, the antibody or antigen binding fragment comprises CDR-H3 of SJ25C1 (e.g., as set forth in SEQ ID NO: 116) and/or CDR-L3 of SJ25C1 (e.g., as set forth in SEQ ID NO: 119). In some embodiments, the antibody or antigen binding fragment comprises CDR-H3 and CDR-L3 of SJ25C1 (e.g., set forth in SEQ ID NOS: 116 and 119, respectively). In some embodiments, the target antibody (which is SJ25C1 or an antibody derived from SJ25C 1) is an antibody or antigen binding sheet Segments comprising one or more of CDR-H1, CDR-H2 and CDR-H3 of SJ25C1 (e.g. as set out in SEQ ID NOS: 114, 115, 116, respectively) and/or one or more of CDR-L1, CDR-L2 and CDR-L3 of SJ25C1 (e.g. as set out in SEQ ID NOS: 117, 118, 119, respectively). In some embodiments, the target antibody (which is SJ25C1 or an antibody derived from SJ25C 1) is an antibody or antigen binding fragment comprising CDR-H1, CDR-H2 and CDR-H3 of SJ25C1 (e.g., set forth in SEQ ID NOS: 114, 115, 116, respectively) and/or CDR-L1, CDR-L2 and CDR-L3 of SJ25C1 (e.g., set forth in SEQ ID NOS: 117, 118, 119, respectively). In some embodiments, the target antibody (which is SJ25C1 or an antibody derived from SJ25C 1) is an antibody or antigen binding fragment comprising CDR-H1, CDR-H2 and CDR-H3 of SJ25C1 (e.g., set forth in SEQ ID NOS: 114, 115, 116, respectively) and CDR-L1, CDR-L2 and CDR-L3 of SJ25C1 (e.g., set forth in SEQ ID NOS: 117, 118, 119, respectively). In some embodiments, the antibody or antigen binding fragment comprises an antigen binding fragment such as an antigen binding fragment (Fab), F (ab ') 2, fab', variable region fragment (Fv), or single chain Fv (scFv). See, e.g., bejcek, B.E. et al (1995) Cancer research.55 (11): 2346-2351.
FMC63 is a mouse monoclonal IgG1 antibody raised against JVM3 cells expressing human CD19 (Nicholson et al (1997): molecular immunology.34 (16-17): 1157-1165). The FMC63 antibody comprises the CDRH1, H2 and H3 sequences set forth in SEQ ID NOS 120-122, respectively, and CDRL1, L2 and L3 sequences set forth in SEQ ID NOS 123-125, respectively. The FMC63 antibody comprises a heavy chain variable region (V) comprising the amino acid sequence SEQ ID NO. 30 H ) And a light chain variable region (V) comprising the amino acid sequence SEQ ID NO. 31 L )。
In some embodiments, the target antibody is FMC63 or an antibody derived from FMC 63. In some embodiments, the antibody derived from FMC63 is V comprising FMC63 H And/or V L An idiotype of FMC63, a paratope of FMC63, or an antibody or antigen binding fragment of one or more Complementarity Determining Regions (CDRs) of FMC 63. In some embodiments, the target antibody (which is FMC63 or an antibody derived from FMC 63) is an antibody or antigen binding fragment comprising V of FMC63 set forth in SEQ ID NO. 30 H Or a variant thereof having at least 90% sequence identity to SEQ ID NO. 30, and/or FMC63 set forth in SEQ ID NO. 31 L Or a variant thereof having at least 90% sequence identity to SEQ ID NO. 31. In some embodiments, the target antibody (which is FMC63 or an antibody derived from FMC 63) is an antibody or antigen binding fragment comprising V of FMC63 set forth in SEQ ID NO. 30 H Or a variant thereof having at least 90% sequence identity to SEQ ID NO. 30, and FMC63 set forth in SEQ ID NO. 31 L Or a variant thereof having at least 90% sequence identity to SEQ ID NO. 31. In some embodiments, the antibody or antigen binding fragment comprises V of FMC63 set forth in SEQ ID NO. 30 and SEQ ID NO. 31, respectively H And V L . In some embodiments, the variant has at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO. 30 and/or SEQ ID NO. 31.
In some embodiments, the target antibody (which is FMC63 or an antibody derived from FMC 63) is an antibody or antigen binding fragment comprising FMC63V set forth in SEQ ID NO. 30 H Is a heavy chain CDR (CDR-H) (such as set forth in SEQ ID NOS: 120-122), and/or FMC63V set forth in SEQ ID NO: 31) L Is a light chain CDR (CDR-L) (such as set forth in SEQ ID NOS: 123-125). In some embodiments, the antibody or antigen binding fragment comprises CDR-H3 of FMC63 (e.g., as set forth in SEQ ID NO: 122) and/or CDR-L3 of FMC63 (e.g., as set forth in SEQ ID NO: 125). In some embodiments, the antibody or antigen binding fragment comprises CDR-H3 of FMC63 (e.g., set forth in SEQ ID NO: 122) and CDR-L3 (e.g., set forth in SEQ ID NO: 125). In some embodiments, the antibody or antigen binding fragment comprises one or more of CDR-H1, CDR-H2 and CDR-H3 of FMC63 (set forth in SEQ ID NOS: 120, 121, 122, respectively) and/or one or more of CDR-L1, CDR-L2 and CDR-L3 of FMC63 (set forth in SEQ ID NOS: 123, 124, 125, respectively). In some embodiments, the antibody or antigen binding fragment comprises CDR-H1, CDR-H2 and CDR-H3 of FMC63 (set forth in SEQ ID NOS: 120, 121, 122, respectively) and/or CDR-L1, CD of FMC63 R-L2 and CDR-L3 (set forth in SEQ ID NOS: 123, 124, 125, respectively). In some embodiments, the antibody or antigen binding fragment comprises CDR-H1, CDR-H2 and CDR-H3 of FMC63 (set forth in SEQ ID NOS: 120, 121, 122, respectively) and CDR-L1, CDR-L2 and CDR-L3 of FMC63 (set forth in SEQ ID NOS: 123, 124, 125, respectively). In some embodiments, the antibody or antigen binding fragment comprises an antigen binding fragment such as an antigen binding fragment (Fab), F (ab ') 2, fab', variable region fragment (Fv), or single chain Fv (scFv).
In some embodiments, the anti-idiotype antibodies provided include antibodies that specifically bind to a variable domain (Fv) such as a single chain Fv (scFv) derived from SJ25C1 or FMC 63. In some embodiments, an anti-idiotype antibody specifically binds to a particular epitope or region of an Fv, which is typically an epitope or region that comprises one or more complementarity determining regions. In some embodiments, the anti-idiotype antibody specifically binds to an overlapping epitope or region of the Fv paratope.
In some embodiments, anti-idiotype antibodies provided include those antibodies that specifically bind to an anti-CD 19 module derived from SJ25C1 or FMC63, which SJ25C1 or FMC63 is included as part of the extracellular domain of a target Chimeric Antigen Receptor (CAR). In some embodiments, the target CAR contains an antigen binding portion that contains an antigen binding fragment or portion of a SJ25C1 or FMC63 antibody molecule or an SJ25C1 or FMC63 antibody. In some embodiments, the target CAR comprises an antigen binding domain that is an scFv derived from VH and VL chains of antibody SJ25C1 or FMC 63. In some embodiments, an anti-idiotype antibody that specifically binds to an anti-CD 19CAR containing scFv derived from antibody SJ25C1 or FMC63 is provided. Exemplary features of the CAR are described further below.
The term "antibody" is used herein in its broadest sense and includes polyclonal and monoclonal antibodies, including whole antibodies and functional (antigen-binding) antibody fragments, including fragment antigen-binding (Fab) fragments, F (ab') 2 Fragments, fab' fragments, fv fragments, recombinant IgG (rIgG) fragments, single chain antibody fragmentsFragments, including single chain variable fragments (scFv) and single domain antibody (e.g., sdAb, sdFv, nanobody) fragments. The term encompasses genetically engineered and/or other modified forms of immunoglobulins, such as intracellular antibodies, peptibodies, chimeric antibodies, fully human antibodies, humanized antibodies and heteroconjugate antibodies, multispecific (e.g., bispecific) antibodies, diabodies, triabodies and tetrabodies, tandem di-scFv, tandem tri-scFv. Unless otherwise indicated, the term "antibody" is to be understood as encompassing functional antibody fragments thereof. The term also encompasses whole or full length antibodies, including antibodies of any type or subtype, including IgG and its subtypes, igM, igE, igA and IgD.
The term "anti-idiotype antibody" refers to an antibody, including antigen-binding fragments thereof, that specifically recognizes, specifically targets, and/or specifically binds to a unique site (such as an antigen-binding fragment) of the antibody. Unique positions of an antibody may include, but are not necessarily limited to, residues within one or more of the Complementarity Determining Regions (CDRs) of the antibody, the variable regions of the antibody, and/or such variable regions and/or incomplete portions or portions of such CDRs, and/or any combination of the foregoing. The CDRs may be one or more selected from the group consisting of: CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3. The variable region of an antibody may be a heavy chain variable region, a light chain variable region, or a combination of a heavy chain variable region and a light chain variable region. The partial fragment or portion of the heavy chain variable region and/or the light chain variable region of an antibody may be a fragment of 2 or more, 5 or more, or 10 or more contiguous amino acids contained within the heavy chain variable region or the light chain variable region of an antibody, e.g., about 2 to about 100, about 5 to about 100, about 10 to about 100, about 2 to about 50, about 5 to about 50, or about 10 to about 50 contiguous amino acids; the unique positions may comprise a plurality of discrete amino acid segments. The partial fragments of the heavy and light chain variable regions of an antibody may be fragments of 2 or more, 5 or more, or 10 or more contiguous amino acids contained within the variable region, e.g., about 2 to about 100, about 5 to about 100, about 10 to about 100, about 2 to about 50, about 5 to about 50, or about 10 to about 50 contiguous amino acids; unique positions can include fragments of multiple discrete amino acids; in some embodiments, one or more CDRs or CDR fragments are contained. CDR fragments may be 2 or more, or 5 or more amino acids, contiguous or non-contiguous within a CDR. Thus, the unique position of an antibody can be from about 2 to about 100, from about 5 to about 100, from about 10 to about 100, from about 2 to about 50, from about 5 to about 50, or from about 10 to about 50 consecutive amino acids comprising one or more CDRs or one or more CDR fragments within the heavy chain variable region or the light chain variable region of the antibody. In another embodiment, the unique position may be a single amino acid located in the variable region of the antibody, e.g., a CDR site.
In some embodiments, the unique position is any single epitope or epitope within the variable portion of the antibody. In some cases, it may overlap with the actual antigen binding site of the antibody, and in some cases, it may comprise a variable region sequence outside the antigen binding site of the antibody. In some embodiments, a group of individual idiotypes of an antibody is referred to as the "idiotype" of such an antibody.
The terms "complementarity determining region" and "CDR" are known in the art as synonymous with "hypervariable region" or "HVR," and refer to non-contiguous amino acid sequences within the variable region of an antibody that confer antigen specificity and/or binding affinity. Typically, there are three CDRs in each heavy chain variable region (CDR-H1, CDR-H2, CDR-H3) and three CDRs in each light chain variable region (CDR-L1, CDR-L2, CDR-L3). "framework regions" and "FR" are known in the art and refer to the non-CDR portions of the heavy and light chain variable regions. Typically, there are four FRs (FR-H1, FR-H2, FR-H3 and FR-H4) in each full-length heavy chain variable region, and four FRs (FR-L1, FR-L2, FR-L3 and FR-L4) in each full-length light chain variable region.
The exact amino acid sequence boundaries for a given CDR or FR can be readily determined using any of a number of well known schemes, including those described by: kabat et Al (1991), "Sequences of Proteins of Immunological Interest," 5 th edition, public Health Service, national Institutes of Health, bethesda, MD ("Kabat" number scheme), al-Lazikani et Al, (1997) JMB 273,927-948 ("Chothia" number scheme), macCallum et Al, J.mol.biol.262:732-745 (1996), "anti-antigen interactions: contact analysis and binding site topography," J.mol.biol.262,732-745 "(" Contact "number scheme), lefranc MP et Al," IMGT unique numbering for immunoglobulin and T cell receptor variable domains and Ig superfamily V-like domains, "Dev Comp Immunol,2003Jan;27 (1) 55-77 ("IMGT" numbering scheme), honygger A and Pluckthun A, "Yet another numbering scheme for immunoglobulin variable domains: an automatic modeling and analysis tool," J Mol Biol,2001Jun 8;309 (3) 657-70, ("Aho" number scheme).
The boundaries of a given CDR or FR may vary depending on the scheme used for identification. For example, the Kabat scheme is based on structural alignment, while the Chothia scheme is based on structural information. Numbering of both Kabat and Chothia protocols is based on the most common antibody region sequence length, with insertions indicated by the insert letter, e.g. "30a", and deletions in some antibodies. Both of these schemes place certain insertions and deletions ("indels") at different locations, resulting in a number difference. The Contact scheme is based on analysis of complex crystal structures and is similar in many respects to the Chothia numbering scheme.
Table 1 below lists exemplary location boundaries for CDR-L1, CDR-L2, CDR-L3 and CDR-H1, CDR-H2, CDR-H3 identified by the Kabat, chothia and Contact schemes, respectively. For CDR-H1, residue numbers are listed using both the Kabat and Chothia numbering schemes. FR is located between CDRs, e.g., where FR-L1 is located between CDR-L1 and CDR-L2, etc. It should be noted that since the illustrated Kabat numbering scheme will be inserted at H35A and H35B, the ends of the Chothia CDR-H1 loop vary between H32 and H34 when numbered using the illustrated Kabat numbering convention, depending on the length of the loop.
TABLE 1
1-Kabat et al (1991), "Sequences of Proteins of Immunological Interest,"5th Ed.Public Health Service,National Institutes of Health,Bethesda,MD
2-Al-Lazikani et Al, (1997) JMB 273,927-948
Thus, unless otherwise indicated, "CDR" or "complementarity determining region" or an individual-designated CDR (e.g., "CDR-H1, DR-H2") of a given antibody or region thereof (such as a variable region thereof) should be understood to include complementarity determining regions (or specificities) defined by any of the foregoing schemes. For example, when a particular CDR (e.g., CDR-H3) is stated to contain a given V H Or V L Where the amino acid sequence of a corresponding CDR in the amino acid sequence is one, it is to be understood that such CDR has the corresponding CDR (e.g., CDR-H3) sequence within the variable region, as defined by any of the schemes described above. In some embodiments, the specified CDR sequences are specified.
Likewise, unless otherwise indicated, the FR or individually specified FR (e.g., FR-H1, FR-H2) of a given antibody or region thereof (such as the variable region thereof) should be understood to include the framework regions defined (or specific) by any known scheme. In some cases, an identification scheme of a particular CDR, FR, or multiple FR or CDRs (such as those defined by Kabat, chothia or Contact methods) is specified. In other cases, specific amino acid sequences of CDRs or FR are given.
The term "variable region" or "variable domain" refers to a domain of an antibody heavy or light chain that relates to binding of an antibody to an antigen. The variable domains of the heavy and light chains of natural antibodies (V respectively H And V L ) Typically having a similar structure, each domain comprises four conserved Framework Regions (FR) and three CDRs. (see, e.g., kindt et al, kuby Immunology,6th ed., W.H. Freeman and Co., p 91 (2007). Individual V) H Or V L The domain may be sufficient to confer antigen binding specificity. In addition, V from antigen-binding antibodies can be used H Or V L The domains are used to isolate antibodies that bind to a particular antigen to screen for complementary V respectively L Or V H Library of domains. See, e.g., portolano et alImmunol.150:880-887 (1993); clarkson et al Nature 352:624-628 (1991).
Wherein the antibody provided is an antibody fragment. An "antibody fragment" refers to a molecule other than an intact antibody that comprises a portion of the intact antibody that binds to an antigen to which the intact antibody binds. Examples of antibody fragments include, but are not limited to Fv, fab, fab ', fab ' -SH, F (ab ') 2 The method comprises the steps of carrying out a first treatment on the surface of the A diabody; a linear antibody; single chain antibody molecules (e.g., scFv); and multispecific antibodies formed from antibody fragments. In particular embodiments, the antibody is a single chain antibody fragment, such as an scFv, comprising a variable heavy chain region and/or a variable light chain region.
A single domain antibody is an antibody fragment comprising all or part of the heavy chain variable domain or all or part of the light chain variable domain of an antibody. In certain embodiments, the single domain antibody is a human single domain antibody.
Antibody fragments may be produced by a variety of techniques, including, but not limited to, proteolytic digestion of intact antibodies and production by recombinant host cells. In some embodiments, the antibody is a recombinantly produced fragment, such as a fragment comprising a non-naturally occurring arrangement, such as a fragment having two or more antibody regions or chains joined by a synthetic linker (e.g., a peptide linker), and/or a fragment that cannot be produced by enzymatic digestion of a naturally occurring intact antibody. In some aspects, the antibody fragment is an scFv.
A "humanized" antibody is one in which all or substantially all CDR amino acid residues are derived from non-human CDRs and all or substantially all Framework Region (FR) amino acid residues are derived from human FRs. In some embodiments, a "humanized" form of a non-human antibody (e.g., a murine antibody) is a chimeric antibody that contains minimal sequences derived from a non-human immunoglobulin. In certain embodiments, the humanized antibody is an antibody from a non-human species that has one or more Complementarity Determining Regions (CDRs) from the non-human species and a Framework Region (FR) from a human immunoglobulin molecule. In some embodiments, the humanized antibody optionally can comprise at least a portion of an antibody constant region derived from a human antibody. "humanized form" of a non-human antibody refers to a variant of a non-human antibody that has undergone humanization, typically in order to reduce immunogenicity to humans, while retaining the specificity and affinity of the parent non-human antibody. In some embodiments, some FR residues in a humanized antibody are substituted with corresponding residues from a non-human antibody (e.g., an antibody from which CDR residues are derived), e.g., for restoring or increasing antibody specificity or affinity. (see, e.g., queen, U.S. Pat. No.5,585,089 and Winter, U.S. Pat. No.5,225,539.) such chimeric and humanized monoclonal antibodies can be produced by recombinant DNA techniques known in the art.
In certain embodiments, the humanized antibody is a human immunoglobulin (recipient antibody) in which residues from the heavy chain variable region of the recipient are replaced with residues from a heavy chain variable region of a non-human species (donor antibody) such as mouse, rat, rabbit, or non-human primate having the desired specificity, affinity, and/or capacity. In some cases, FR residues of the human immunoglobulin are replaced with corresponding non-human residues. In addition, the humanized antibody may comprise residues not found in the recipient antibody or in the donor antibody. In some embodiments, the nucleic acid sequences encoding the human variable heavy and variable light chains are altered to replace one or more CDR sequences of the human (acceptor) sequence with sequences encoding corresponding CDRs in the non-human antibody sequence (donor sequence). In some embodiments, the human receptor sequence may comprise FR derived from a different gene. In a specific embodiment, the humanized antibody will comprise substantially all of at least one (and typically two) variable domain, in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin and all or substantially all of the FR is that of a human immunoglobulin sequence. In some embodiments, the humanized antibody optionally will also comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For more details, see, e.g., jones et al, nature 321:522-525 (1986); riechmann et al Nature332:323-329 (1988); and Presta, curr.Op.struct.biol.2:593-596 (1992). See also, e.g., vaswani and Hamilton, ann. Allergy, asthma & Immunol.1:105-115 (1998); harris, biochem. Soc. Transactions 23:1035-1038 (1995); hurle and Gross, curr.op.Biotech.5:428-433 (1994); and U.S. patent nos. 6,982,321 and 7,087,409, which are incorporated herein by reference. In some embodiments, provided herein are humanized anti-idiotype antibodies.
In particular embodiments, the antibody, e.g., an anti-idiotype antibody, is humanized. In certain embodiments, the antibodies are humanized by any suitable known means. For example, in some embodiments, a humanized antibody may have one or more amino acid residues introduced into it from a non-human source. These non-human amino acid residues are commonly referred to as "import" amino acid residues, and are typically taken from an "import" variable domain. In particular embodiments, humanization may be performed substantially by following the method of Winter and colleagues (Jones et al (1986) Nature 321:522-525; riechmann et al (1988) Nature 332:323-327; verhoeyen et al (1988) Science 239:1534-1536), such as by substituting hypervariable region sequences for the corresponding sequences of human antibodies. Thus, such "humanized" antibodies are chimeric antibodies (U.S. Pat. No.4,816,567) in which substantially less complete human variable domains have been replaced by corresponding sequences from non-human species. In certain embodiments, the humanized antibody is a human antibody in which some hypervariable region residues and possibly some FR residues are substituted with residues from a similar site in a rodent antibody.
Sequences encoding full length antibodies can then be obtained by ligating the provided variable heavy and variable light chain sequences to human constant heavy and constant light chain regions. Suitable human constant light chain sequences include kappa and lambda constant light chain sequences. Suitable human constant heavy chain sequences include IgG1, igG2, and sequences encoding IgG1 mutants, which are provided with immunostimulatory properties. Such mutants may have reduced ability to activate complement and/or antibody dependent cytotoxicity and are described in U.S. Pat. nos. 5,624,821; WO 99/58372, U.S. Pat. No.6,737,056. Suitable constant heavy chains also include deleted IgG1 comprising substitutions E233P, L234V, L235A, A327G, A330S, P331S and residue 236. In another embodiment, the full length antibody comprises IgA, igD, igE, igM, igY or IgW sequences.
Suitable human donor sequences may be determined by comparing the peptide sequences encoded by the mouse donor sequences with sequences of a set of human sequences, preferably sequences encoded by human germline immunoglobulin genes or mature antibody genes. Human sequences with high sequence homology, preferably with the highest homology determined, can be used as acceptor sequences for the humanization process.
In addition to exchanging human CDRs for mouse CDRs, further manipulations in human donor sequences can be performed to obtain sequences encoding humanized antibodies with optimized properties (e.g., antigen affinity).
In addition, the altered human acceptor antibody variable domain sequences may also be made to encode one or more amino acids corresponding to positions 4, 35, 38, 43, 44, 46, 58, 62, 64, 65, 66, 67, 68, 69, 73, 85, 98 of the light chain variable region and positions 2, 4, 36, 39, 43, 45, 69, 70, 74, 75, 76, 78, 92 of the heavy chain variable region of the non-human donor sequence (according to the Kabat numbering system) (Carter and Presta, U.S. patent No.6,407,213).
In particular embodiments, it is generally desirable to humanize antibodies while retaining high affinity for antigens and other advantageous biological properties. To achieve this goal, in some embodiments, humanized antibodies are prepared by a process of analyzing a parent sequence and various conceptual humanized products using a three-dimensional model of the parent and humanized sequences. Three-dimensional immunoglobulin models are generally available and familiar to those skilled in the art. A computer program is available that illustrates and displays the possible three-dimensional conformational structure of the selected candidate immunoglobulin sequence. Examination of these displays allows analysis of the likely role of residues in the function of the candidate immunoglobulin sequence, i.e., analysis of residues that affect the ability of the candidate immunoglobulin to bind its antigen. In this way, FR residues can be selected and combined according to the receptor sequence and the input sequence such that desired antibody characteristics, such as increased affinity for the target antigen, are achieved. Typically, hypervariable region residues are directly and predominantly involved in influencing antigen binding.
In particular embodiments, the selection of human variable domains (both light and heavy chains) for use in making humanized antibodies may be important for reducing antigenicity. The sequence of the rodent antibody variable domain is screened against a whole library of known human variable domain sequences according to the so-called "best fit" method. The human sequence closest to the rodent is then accepted as the human framework for the humanized antibody. See, e.g., sims et al (1993) J.Immunol.151:2296; chothia et al (1987) J.mol.biol.196:901. Another approach uses a specific framework of consensus sequences of all human antibodies derived from a specific light chain or heavy chain subgroup. The same framework can be used for several different humanized antibodies. See, e.g., carter et al (1992) Proc. Natl. Acad. Sci. USA,89:4285; presta et al (1993) J.Immunol.,151:2623.
Wherein the antibody provided is a human antibody. A "human antibody" is an antibody having an amino acid sequence that corresponds to an amino acid sequence of an antibody produced by a human or human cell, or an antibody produced by a non-human source (including a human antibody library) using a human antibody repertoire or other human antibody coding sequence. The term excludes humanized versions of non-human antibodies that comprise non-human antigen binding regions, such as those antibodies in which all or substantially all of the CDRs are non-human.
Human antibodies can be prepared by administering an immunogen to a transgenic animal that has been modified to produce whole human antibodies or whole antibodies having human variable regions responsive to antigen challenge. Such animals typically contain all or part of the human immunoglobulin loci, either in place of endogenous immunoglobulin loci, or extrachromosomally present or randomly integrated into the animal chromosome. In such transgenic animals, the endogenous immunoglobulin loci have typically been inactivated. Human antibodies can also be derived from human antibody libraries, including phage display libraries and cell-free libraries, which contain antibody coding sequences derived from human repertoires.
Wherein the antibodies provided are monoclonal antibodies, including monoclonal antibody fragments. The term "monoclonal antibody" as used herein refers to an antibody obtained from or within a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical, except that variants may exist that contain naturally occurring mutations or are produced during production of a monoclonal antibody preparation, such variants typically being present in minor amounts. In contrast to polyclonal antibody preparations, which typically include different antibodies directed against different epitopes, each monoclonal antibody of a monoclonal antibody preparation is directed against a single epitope on the antigen. The term should not be construed as requiring the production of antibodies by any particular method. Monoclonal antibodies can be made by a variety of techniques including, but not limited to, production from hybridomas, recombinant DNA methods, phage display, and other antibody display methods.
SJ25C 1-derived antibodies
In some embodiments, an anti-idiotype antibody specific for a target anti-CD 19 antibody is provided, which is or is derived from antibody SJ25C1 or an antigen binding fragment thereof. In some embodiments, the provided antibodies or antigen binding fragments are specific for scFv derived from SJ25C 1.
In some embodiments, a polypeptide comprising a heavy chain variable (V H ) Anti-idiotype antibodies or antigen-binding fragments thereof, the heavy chain variable (V H ) Region and V set forth in SEQ ID NO. 1 H The region amino acid sequence has at least 90% sequence identity, such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto.
In some embodiments, an anti-idiotype antibody or antigen-binding fragment thereof is provided that includes a heavy chain Variable (VH) region containing heavy chain complementarity determining region 3 (CDR-H3) having an amino acid sequence set forth in SEQ ID No. 11 or 84 and/or comprising a heavy chain variable (V) set forth in SEQ ID No. 1 H ) CDR-H3 within the sequence.
In some of any such embodiments, V H The region comprises heavy chain complementarity determining region 1 (CDR-H1) comprising the amino acid sequences set forth in SEQ ID NO. 9, 78, 79 or 80 and/or V set forth in SEQ ID NO. 1 H CDR-H1 within the sequence; and/orHeavy chain complementarity determining region 2 (CDR-H2) comprising the amino acid sequences set forth in SEQ ID NO. 10, 81, 82 or 83 and/or V comprising the amino acid sequences set forth in SEQ ID NO. 1 H CDR-H2 within the sequence.
An anti-idiotype antibody or antigen-binding fragment thereof is provided comprising a heavy chain Variable (VH) region comprising heavy chain complementarity determining region 1 (CDR-H1), CDR-H2, and CDR-H3, wherein CDR-H1 comprises the amino acid sequence set forth in SEQ ID NOs 9, 78, 79, or 80; CDR-H2 comprises the amino acid sequence set forth in SEQ ID NO. 10, 81, 82 or 83; and/or CDR-H3 comprises the amino acid sequence set forth in SEQ ID NO. 11 or 84. In some embodiments, antibodies or antigen binding fragments thereof are provided that include CDR-H1 having the amino acid sequences set forth in SEQ ID NO 9, 78, 79 or 80; CDR-H2 having the amino acid sequence set forth in SEQ ID NO 10, 81, 82 or 83; and CDR-H3 having the amino acid sequence set forth in SEQ ID NO 11 or 84.
An anti-idiotype antibody or antigen-binding fragment thereof is provided comprising heavy chain complementarity determining regions 1 (CDR-H1), CDR-H2 and CDR-H3, each comprising the three complementarity determining regions set forth in SEQ ID NO. 1, comprising V H Amino acid sequences of CDR-H1, CDR-H2 and CDR-H3 within the amino acid sequences of the regions.
In some of any such embodiments, V H The region contains the framework region 1 (FR 1), FR2, FR3 and/or FR4 sequence that has at least 90% sequence identity to the FR1, FR2, FR3 and/or FR4, respectively, of the amino acid sequence set forth in SEQ ID NO. 1. In some embodiments, V H The region contains the framework region 1 (FR 1), FR2, FR3 and/or FR4 sequence that has at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to the FR1, FR2, FR3 and/or FR4, respectively, of the amino acid sequence set forth in SEQ ID NO. 1. In some embodiments, V H The region contains the framework region 1 (FR 1), FR2, FR3 and FR4 sequences that have at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to the FR1, FR2, FR3 and FR4, respectively, of the amino acid sequence set forth in SEQ ID NO. 1.
At any placeIn some of these embodiments, V H The region has the amino acid sequence set forth in SEQ ID NO. 1.
In some of any such embodiments, the anti-idiotype antibody or antigen-binding fragment is heavy chain only, VH only, and/or does not include VL or antigen-binding portion thereof, and/or the antigen-binding site of the anti-idiotype antibody or fragment includes only residues from a heavy chain and/or does not include residues from a light chain.
In some of any such embodiments, the anti-idiotype antibody or fragment is free of light chain variable (V L ) Regions which do not contain CDR-L1, CDR-L2 and/or CDR-L3 and/or which contain only V H Single domain antibodies (sdabs) of the region. In some embodiments, the antibody or fragment is an sdAb comprising only VH regions from any of the described VH regions.
In some embodiments of any anti-idiotype antibody or fragment comprising any of the above VH region sequences, the anti-idiotype antibody or fragment further comprises a light chain variable (V L ) A zone. In some such embodiments, V L Region and V set forth in SEQ ID NO. 5 L The region amino acid sequence has at least 90% sequence identity, such as with V set forth in SEQ ID NO. 5 L The region amino acid sequence has at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity.
In some of any such embodiments, V L The region comprises light chain complementarity determining region 3 (CDR-L3), said CDR-L3 comprising the amino acid sequence set forth in SEQ ID NO. 14 or 87. In some of any such embodiments, the VL region comprises a light chain complementarity determining region 3 (CDR-L3) having the amino acid sequence set forth in SEQ ID NO 14 or 87.
In some of any such embodiments, V L The region comprises light chain complementarity determining region 1 (CDR-L1) comprising the amino acid sequence set forth in SEQ ID NO. 12 or 85 and/or V comprising the amino acid sequence set forth in SEQ ID NO. 5 L CDR-L1 within the sequence; and/or light chain complementarity determining region 2 (CDR-L2) comprising the amino acid sequence set forth in SEQ ID NO. 13 or 86 and/or V comprising the amino acid sequence set forth in SEQ ID NO. 5 L CDR-L2 within the sequence. In some of any such embodiments, V L The region comprises light chain complementarity determining region 1 (CDR-L1) having the amino acid sequence set forth in SEQ ID NO. 12 or 85 and/or comprises V set forth in SEQ ID NO. 5 L CDR-L1 within the sequence; and/or light chain complementarity determining region 2 (CDR-L2) having the amino acid sequence set forth in SEQ ID NO. 13 or 86 and/or comprising V set forth in SEQ ID NO. 5 L CDR-L2 within the sequence.
In some of any such embodiments, V L The region comprises CDR-L1 comprising the amino acid sequence set forth in SEQ ID NO 12 or 85; CDR-L2 comprising the amino acid sequence set forth in SEQ ID NO 13 or 86; and CDR-L3 comprising the amino acid sequence set forth in SEQ ID NO 14 or 57.
In some of any such embodiments, V L The regions comprise CDR-L1, CDR-L2 and CDR-L3, which comprise V as set forth in SEQ ID NO 5, respectively L Amino acid sequences of CDR-L1, CDR-L2 and CDR-L3 within the amino acid sequences of the regions.
In some of any such embodiments, V L The region comprises framework region 1 (FR 1), FR2, FR3 and/or FR4 that has at least 90% sequence identity with FR1, FR2, FR3 and/or FR4, respectively, of the amino acid sequence set forth in SEQ ID NO. 5. In some embodiments, V L The region comprises the framework region 1 (FR 1), FR2, FR3 and/or FR4 sequence that has at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to the FR1, FR2, FR3 and/or FR4, respectively, of the amino acid sequence set forth in SEQ ID NO. 5. In some embodiments, V L The regions comprise the framework region 1 (FR 1), FR2, FR3 and FR4 sequences that have at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to the FR1, FR2, FR3 and FR4, respectively, of the amino acid sequence set forth in SEQ ID NO. 5.
In some of any such embodiments, V L The region has the amino acid sequence set forth in SEQ ID NO. 5.
An anti-idiotype antibody or antigen-binding fragment thereof is provided which is comprised in SEQV listed in ID NO. 1 H Amino acid sequences of CDR-H1, CDR-H2 and CDR-H3 sequences contained within the amino acid sequences of the regions; and/or amino acid sequences comprising CDR-L1, CDR-L2 and CDR-L3 sequences contained within the light chain Variable (VL) region amino acid sequences set forth in SEQ ID NO. 5.
Is provided with V H And V L Anti-idiotype antibodies or antigen-binding fragments thereof, of region V H And V L The regions have amino acid sequences having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NOs 1 and 5, respectively.
In some embodiments, a polypeptide comprising a polypeptide having the amino acid sequences set forth in SEQ ID NOs 1 and 5, respectively, is provided H And V L An anti-idiotype antibody or antigen-binding fragment of a region.
In some of any such embodiments, V H And V L The amino acid sequences of SEQ ID NOs 1 and 5 are included, respectively.
In some embodiments, the anti-idiotype antibody specific for antibody SJ25C1 or antigen binding fragment thereof is a single chain antibody fragment, such as scFv or diabody. In some embodiments, single chain antibodies include a linkage of two antibody domains or regions such as variable heavy chains (V H ) Region and variable light chain (V L ) Is provided for the connection of the one or more connectors. The linker is typically a peptide linker, such as a flexible and/or soluble peptide linker. Wherein the linker is a glycine and serine-rich and/or in some cases threonine-rich linker. In some embodiments, the linker further comprises charged residues, such as lysine and/or glutamic acid, which may increase solubility. In some embodiments, the linker further comprises one or more prolines.
In some embodiments, the anti-idiotype antibody is an intact antibody or a full length antibody. In some embodiments, the anti-ID may contain at least a portion of an immunoglobulin constant region, such as one or more constant region domains. In some embodiments, the constant regions comprise a light chain constant region (CL) and/or a heavy chain constant region 1 (CH 1). In some embodiments, the anti-ID comprises a CH2 and/or CH3 domain, such as an Fc region. In some embodiments, the Fc region is that of a human IgG (such as IgG1 or IgG 4). In some embodiments, the anti-idiotype antibody contains a CH domain set forth in SEQ ID NO. 2, or a portion thereof, or an amino acid sequence exhibiting at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO. 2. In some embodiments, the anti-idiotype antibody contains the CL domain set forth in SEQ ID No. 6 or a portion thereof, or an amino acid sequence or portion thereof that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID No. 6.
In some embodiments, an anti-idiotype antibody specific for SJ25C1 comprises a heavy chain sequence set forth in SEQ ID No. 3 or a sequence exhibiting at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID No. 3 and/or comprises a light chain sequence set forth in SEQ ID No. 7 or a sequence exhibiting at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID No. 7. In some embodiments, an anti-idiotype antibody specific for SJ25C1 comprises the heavy chain sequence set forth in SEQ ID NO. 3 and/or the light chain sequence set forth in SEQ ID NO. 7. In some embodiments, the heavy and/or light chain of the anti-idiotype antibody further comprises a signal peptide. In some cases, the signal peptide has the sequence set forth in SEQ ID NO. 4 or SEQ ID NO. 8.
In some embodiments, the anti-idiotype antibody is an antigen binding fragment. In some embodiments, the antigen binding fragment is selected from the group consisting of: fragment antigen binding (Fab) fragments, F (ab') 2 Fragments, fab' fragments, fv fragments, single chain variable fragments (scFv), or single domain antibodies.
Thus, single chain antibody fragments, such as scFv and diabodies, particularly human single chain fragments, are provided which are universalOften comprising linking two anti-idiotype antibody domains or regions such as V H And V L A linker of the domain. The linker is typically a peptide linker, e.g. a flexible and/or soluble peptide linker, such as a glycine and serine rich peptide linker.
In some aspects, the glycine and serine (and/or threonine) rich linker comprises at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of such amino acids. In some embodiments, they comprise at least or at least about 50%, 55%, 60%, 70%, or 75% glycine, serine, and/or threonine. In some embodiments, the linker consists essentially entirely of glycine, serine, and/or threonine. The linker length is typically between about 5 to about 50 amino acids, typically between 10 or about 10 to 30 or about 30, e.g., 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30, and in some examples, between 10 to 25 amino acids in length. Exemplary linkers include linkers having sequences GGGS (3 GS; SEQ ID NO: 29) or GGGGS (4 GS; SEQ ID NO: 26) with different numbers of repeats, such as 2, 3, 4, and 5 repeats of such sequences. Exemplary linkers include linkers having or consisting of the sequences set forth in SEQ ID NO. 25 (GGGGSGGGGSGGGGS). Exemplary linkers further include linkers having or consisting of the sequences set forth in SEQ ID NO. 33 (GSTSGSGKPGSGEGSTKG).
In some embodiments, the anti-idiotype antibody comprises an isolated antibody. In some embodiments, the anti-ID is humanized, recombinant, and/or monoclonal. In some embodiments, the anti-ID is a human anti-ID.
In some embodiments, the anti-idiotype antibody specific for SJ25C1 is humanized. In particular embodiments, all or substantially all CDR amino acid residues of a humanized anti-idiotype antibody specific for SJ25C1 are derived from anti-SJ 25C1 non-human CDRs. In some embodiments, the humanized anti-idiotype antibody specific for SJ25C1 comprises at least a portion of an antibody constant region derived from a human antibody.
In certain embodiments, humanized anti-idiotype antibodies specific for SJ25C1 comprise human immunoglobulins (recipient antibody) in which residues from the heavy chain variable region of the recipient are replaced by residues from the heavy chain variable region of a non-human anti-idiotype antibody specific for SJ25C 1. In some cases, FR residues of the human immunoglobulin are replaced with corresponding non-human residues. In some embodiments, the humanized antibodies contain FR derived from a different gene. In some embodiments, the humanized anti-idiotype antibody specific for SJ25C1 comprises at least a portion of an immunoglobulin constant region (Fc), typically a constant region of a human immunoglobulin.
In some embodiments, the humanized anti-idiotype antibody specific for SJ25C1 contains an altered human acceptor antibody variable domain sequence that has been made to encode one or more amino acid residues corresponding to positions 4, 35, 38, 43, 44, 46, 58, 62, 64, 65, 66, 67, 68, 69, 73, 85, 98 (Kabat) and position 2, 4, 36, 39, 43, 45, 69, 70, 74, 75, 76, 78, 92 (Kabat) of the light chain variable region of the non-human donor sequence.
In certain embodiments, the anti-idiotype antibody specific for SJ25C1 is humanized. In specific embodiments, the humanized anti-idiotype antibody specific for SJ25C1 contains one or more of the CDR-L1, CDR-L2, CDR-L3 and CDR-H1, CDR-H2, CDR-H3 regions of a non-human anti-idiotype antibody specific for SJ25C 1. In some embodiments, some or all of the CDR-L1, CDR-L2, CDR-L3 and CDR-H1, CDR-H2, CDR-H3 regions contain one or more amino acid modifications. In some embodiments, the modification replaces a non-human amino acid residue with a human residue. In specific embodiments, one or more of CDR-L1, CDR-L2, CDR-L3 and CDR-H1, CDR-H2, CDR-H3 are inserted into the FR region of a human antibody. In a specific embodiment, CDR-L1, CDR-L2, CDR-L3 and CDR-H1, CDR-H2, CDR-H3 of the non-human anti-idiotype antibody are CDRs of the VH and VL regions having the amino acid sequences set forth in SEQ ID NOS 1 and 5, respectively. In some embodiments, all of the CDRs-L1, CDR-L2, CDR-L3 and CDR-H1, CDR-H2, CDR-H3 of the anti-idiotype antibody specific for SJ25C1 are inserted into the FRs of a human antibody. In specific embodiments, the CDR and FR regions are regions identified by Kabat, chothia, abM and/or Contact protocols.
In specific embodiments, one or more or all of CDR-L1, CDR-L2, CDR-L3 and CDR-H1, CDR-H2, CDR-H3 of a non-human anti-idiotype antibody specific for SJ25C1 are inserted into the framework region of a human antibody. In some embodiments, the human antibodies are IgA, igD, igE, igG and IgM antibodies. In particular embodiments, the human antibody is one of the subclasses of human IgA, igD, igE, igG and IgM, e.g., human IgG 1 、IgG 2 、IgG 3 、IgG 4 、IgA 1 Or IgA 2 . In some embodiments, one or more or all of CDR-L1, CDR-L2, CDR-L3 and CDR-H1, CDR-H2, CDR-H3 of a non-human anti-idiotype antibody specific for SJ25C1 is inserted into a framework region from and/or derived from an antigen binding region of a human antibody. In certain embodiments, the antigen binding fragment is from and/or derived from human IgA, igD, igE, igG and IgM antibodies. The subunit structure and three-dimensional configuration of different classes of human immunoglobulins are well known and are for example generally described by Abbas et al in Cellular and mol.immunology,4th ed. (w.b.samundrs, co., 2000). In some embodiments, the human antibody or antigen binding fragment thereof may be part of a larger fusion molecule formed by covalent or non-covalent binding of the human antibody to one or more other proteins or peptides.
In some embodiments, one or more or all of CDR-L1, CDR-L2, CDR-L3 and CDR-H1, CDR-H2, CDR-H3 of a non-human anti-idiotype antibody specific for SJ25C1 is inserted into a framework region of a human antibody or antigen binding fragment thereof that has all or part of an Fc region. In certain embodiments, the humanized anti-idiotype antibody specific for SJ25C1 contains all or part of the Fc region. In some embodiments, the Fc region has one or more modifications, such as amino acid modifications (e.g., substitutions, insertions, or deletions) at one or more amino acid positions. For example, such modifications may be made to improve half-life, alter binding to one or more types of Fc receptors, and/or alter effector function. In some embodiments, the modified Fc region alters (e.g., reduces) binding to the fcαr relative to binding of the unmodified Fc region to the fcαr. In certain embodiments, the humanized anti-idiotype antibody comprises all or a portion of a modified Fc region having altered (e.g., reduced) binding to an Fc receptor relative to the binding of an unmodified Fc region to an Fc receptor. Non-limiting examples of Fc modifications that alter binding to Fc receptors are described, for example, in U.S. patent nos. 7,217,797 and 7,732,570; and U.S. application nos. US 2010/0143254 and 2010/0143254.
In certain embodiments, a humanized anti-idiotype antibody specific for SJ25C1 has a heavy chain comprising the amino acid sequence set forth in SEQ ID NO. 132 and a light chain comprising the amino acid sequence set forth in SEQ ID NO. 133. In a specific embodiment, the humanized anti-idiotype antibody specific for SJ25C1 has a heavy chain comprising the amino acid sequence set forth in SEQ ID NO. 134 and a light chain comprising the amino acid sequence set forth in SEQ ID NO. 135. In some embodiments, a humanized anti-idiotype antibody specific for SJ25C1 has a heavy chain comprising the amino acid sequence set forth in SEQ ID NO. 136 and a light chain comprising the amino acid sequence set forth in SEQ ID NO. 137.
FMC 63-derived antibodies
In some embodiments, anti-idiotype antibodies specific for a target anti-CD 19 antibody that is or is derived from antibody FMC63 or an antigen binding fragment thereof are provided. In some embodiments, the provided antibodies or antigen binding fragments are specific for scFv derived from FMC 63.
In some embodiments, a polypeptide comprising a heavy chain variable (V H ) Anti-idiotype antibodies or antigen-binding fragments thereof, the heavy chain variable (V H ) Region and V set forth in SEQ ID NO 36 or 58 H The region amino acid sequence has at least 90% sequence identity, such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto.
In some embodiments, antibodies or antigen-binding fragments thereof are provided that include a VH region having: containing ammoniaThe amino acid sequence GYX 3 FX 5 X 6 YX 8 MX 10 Heavy chain complementarity determining region 1 (CDR-H1) (SEQ ID NO: 108), wherein X 3 Is T or S, X 5 Is T or S, X 6 Is D or R, X 8 Is Y or W, and X 10 Is K or N; and/or contain the amino acid sequence WIGX 4 IX 6 PX 8 X 9 X 1 0 X 11 TX 13 X 14 NQX 17 FKX 20 Heavy chain complementarity determining region 2 (CDR-H2) of (SEQ ID NO: 109), wherein X 4 Is D or M, X 6 Is N or H, X 8 Is N or S, X 9 Is N or D, X 10 Is G or S, X 11 Is G or E, X 13 Is D or R, X 14 Y or L, X 17 Is N or K, and X 20 Is G or D; and/or contains the amino acid sequence AX 2 X 3 X 4 X 5 X 6 X 7 X 8 X 9 X 10 X 11 X 12 X 13 X 14 X 15 Heavy chain complementarity determining region 3 (CDR-H3) of (SEQ ID NO: 110), wherein X 2 Is R or S, X 3 Is E or I, X 4 Is G or Y, X 5 Is N or Y, X 6 Is N or E, X 7 Is Y or null, X 8 Is G or null, X 9 Is S or null, X 10 R is null or null, X 11 Is D or null, X 12 Is A or null value, X 13 Is M or null, X 14 Is D or E, and X 15 Is Y or A.
In some embodiments, an anti-idiotype antibody or antigen-binding fragment thereof is provided that includes a heavy chain Variable (VH) region containing heavy chain complementarity determining region 3 (CDR-H3) having an amino acid sequence set forth in SEQ ID NOs 46, 67, 94 or 104 and/or comprising a heavy chain variable (V) set forth in SEQ ID NOs 36 or 58 H ) CDR-H3 within the sequence.
In some of any such embodiments, V H The region comprises heavy chain complementarity determining region 1 (CDR-H1) comprising the amino acid sequences set forth in SEQ ID NO 44, 65, 88, 89, 90, 98, 99 or 100 and/or comprising the amino acid sequences set forth in SEQ ID NO 36 or 58V H CDR-H1 within the sequence; and/or heavy chain complementarity determining region 2 (CDR-H2) comprising the amino acid sequences set forth in SEQ ID NO 45, 66, 91, 92, 93, 101, 102 or 103 and/or V set forth in SEQ ID NO 36 or 58 H CDR-H2 within the sequence.
An anti-idiotype antibody or antigen-binding fragment thereof is provided comprising a heavy chain Variable (VH) region comprising heavy chain complementarity determining region 1 (CDR-H1), CDR-H2, and CDR-H3, wherein CDR-H1 comprises the amino acid sequence set forth in SEQ ID NOs 44, 65, 88, 89, 90, 98, 99, or 100; CDR-H2 comprises the amino acid sequence set forth in SEQ ID NO. 45, 66, 91, 92, 93, 101, 102 or 103; and/or CDR-H3 comprises the amino acid sequence set forth in SEQ ID NO 46, 67, 94 or 104. In some embodiments, antibodies or antigen binding fragments thereof are provided that include CDR-H1 having the amino acid sequences set forth in SEQ ID NO 44, 65, 88, 89, 90, 98, 99 or 100; CDR-H2 having the amino acid sequence set forth in SEQ ID NO. 45, 66, 91, 92, 93, 101, 102 or 103; and CDR-H3 having the amino acid sequence set forth in SEQ ID NO 46, 67, 94 or 104.
An anti-idiotype antibody or antigen-binding fragment thereof is provided comprising heavy chain complementarity determining regions 1 (CDR-H1), CDR-H2 and CDR-H3, said three complementarity determining regions comprising the amino acids set forth in SEQ ID NOS: 36 or 58, respectively, comprising V H Amino acid sequences of CDR-H1, CDR-H2 and CDR-H3 within the amino acid sequences of the regions.
In some embodiments, the anti-idiotype antibody or antigen-binding fragment thereof comprises CDR-H1 as set forth in SEQ ID NO 44, 88, 89 or 90; CDR-H2 as set forth in SEQ ID NO 45, 91, 92 or 93; and/or CDR-H3 as set forth in SEQ ID NO 46 or 94. In some embodiments, the anti-idiotype antibody or antigen-binding fragment thereof comprises CDR-H1 set forth in SEQ ID NO. 65, 98, 99 or 100, respectively; CDR-H2 as set forth in SEQ ID NO 66, 101, 102 or 103; and/or CDR-H3 as set forth in SEQ ID NO 67 or 104.
In some of any such embodiments, V H The region contains the sequences of the framework regions 1 (FR 1), FR2, FR3 and/or FR4, said sequences corresponding to SEQ ID NO. 36 or 58, respectivelyThe FR1, FR2, FR3 and/or FR4 of the listed amino acid sequences have at least 90% sequence identity. In some embodiments, V H The region contains the framework region 1 (FR 1), FR2, FR3 and/or FR4 sequence that has at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to the FR1, FR2, FR3 and/or FR4, respectively, of the amino acid sequence set forth in SEQ ID NO. 36 or 58. In some embodiments, V H The region contains the framework region 1 (FR 1), FR2, FR3 and FR4 sequences that have at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to the FR1, FR2, FR3 and FR4, respectively, of the amino acid sequence set forth in SEQ ID NO. 36 or 58.
In some of any such embodiments, V H The region has the amino acid sequence set forth in SEQ ID NO. 36 or 58.
In some of any such embodiments, the anti-idiotype antibody or antigen-binding fragment is heavy chain only, VH only, and/or does not include VL or antigen-binding portion thereof, and/or the antigen-binding site of the anti-idiotype antibody or fragment includes only residues from a heavy chain and/or does not include residues from a light chain.
In some of any such embodiments, the anti-idiotype antibody or fragment is free of light chain variable (V L ) Regions which do not contain CDR-L1, CDR-L2 and/or CDR-L3 and/or which contain only V H Single domain antibodies (sdabs) of the region. In some embodiments, the antibody or fragment is an sdAb comprising only VH regions from any of the described VH regions.
In some embodiments of any anti-idiotype antibody or fragment comprising any of the above VH region sequences, the anti-idiotype antibody or fragment further comprises a light chain variable (V L ) A zone. In some such embodiments, V L Region and V set forth in SEQ ID NO. 40 or 62 L The region amino acid sequence has at least 90% sequence identity, such as with V set forth in SEQ ID NO. 40 or 62 L The region amino acid sequence has at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity.
In some implementationsIn embodiments, antibodies or antigen-binding fragments thereof are provided that include a VH region having: comprising the amino acid sequence X 1 AX 3 X 4 X 5 X 6 X 7 X 8 YX 10 X 11 Light chain complementarity determining region 1 (CDR-L1) of WY (SEQ ID NO: 111), wherein X 1 Is S or R, X 3 Is S or R, X 4 Is S or G, X 5 Is G or N, X 6 Is V or I, X 7 Is I or H, X 8 Is N or null, X 10 Is M or L, and X 11 Y or A; and/or contain the amino acid sequence X 1 X 2 X 3 YX 5 X 6 X 7 X 8 LAX 11 Light chain complementarity determining region 2 (CDR-L2) of (SEQ ID NO: 112), wherein X 1 Is P or L, X 2 Is W or L, X 3 Is I or V, X 5 Is L or N, X 6 Is T or A, X 7 Is S or K, X 8 Is N or T, and X 11 Is S or D; and/or contain the amino acid sequence QX 2 X 3 X 4 X 5 X 6 PX 8 T (SEQ ID NO: 113) light chain complementarity determining region 3 (CDR-L3), wherein X 2 Is Q or H, X 3 Is W or F, X 4 Is S or W, X 5 Is S or W, X 6 Is N or T, and X 8 Is L or Y.
In some of any such embodiments, V L The region comprises light chain complementarity determining region 3 (CDR-L3), said CDR-L3 comprising the amino acid sequence set forth in SEQ ID NO. 49, 97, 70 or 107. In some of any such embodiments, V L The region comprises light chain complementarity determining region 3 (CDR-L3), said CDR-L3 having the amino acid sequences set forth in SEQ ID NO. 49, 97, 70 or 107.
In some of any such embodiments, V L The region comprises light chain complementarity determining region 1 (CDR-L1) comprising the amino acid sequence set forth in SEQ ID NO. 47, 68, 95 or 105 and/or V comprising the amino acid sequence set forth in SEQ ID NO. 40 or 62 L CDR-L1 within the sequence; and/or light chain complementarity determining region 2 (CDR-L2) comprising the amino acid sequences set forth in SEQ ID NO. 48, 69, 96 or 106 and/or comprising the amino acid sequences set forth in SEQ ID NO. 40 or 62V of (2) L CDR-L2 within the sequence. In some of any such embodiments, V L The region comprises light chain complementarity determining region 1 (CDR-L1) having the amino acid sequences set forth in SEQ ID NO. 47, 68, 95 or 105 and/or comprising V set forth in SEQ ID NO. 40 or 62 L CDR-L1 within the sequence; and/or light chain complementarity determining region 2 (CDR-L2) having the amino acid sequences set forth in SEQ ID NO. 48, 69, 96 or 106 and/or comprising V set forth in SEQ ID NO. 40 or 62 L CDR-L2 within the sequence.
In some of any such embodiments, V L The region comprises CDR-L1 comprising the amino acid sequence set forth in SEQ ID NO. 47, 68, 95 or 105; CDR-L2 comprising the amino acid sequence set forth in SEQ ID NO 48, 69, 96 or 106; and CDR-L3 comprising the amino acid sequence set forth in SEQ ID NO 49, 97, 70 or 107.
In some of any such embodiments, V L The regions comprise CDR-L1, CDR-L2 and CDR-L3 comprising V as set forth in SEQ ID NO 40 or 62, respectively L Amino acid sequences of CDR-L1, CDR-L2 and CDR-L3 within the amino acid sequences of the regions.
In some embodiments, the anti-idiotype antibody or antigen-binding fragment thereof comprises CDR-L1 set forth in SEQ ID NO. 47 or 95; CDR-L2 as set forth in SEQ ID NO 48 or 96; and/or CDR-L3 as set forth in SEQ ID NO 49 or 97. In some embodiments, the anti-idiotype antibody or antigen-binding fragment thereof comprises CDR-L1 set forth in SEQ ID NO 68 or 105, respectively; CDR-L2 as set forth in SEQ ID NO 69 or 106; and/or CDR-L3 as set forth in SEQ ID NO 70 or 107.
In some of any such embodiments, V L The region comprises framework region 1 (FR 1), FR2, FR3 and/or FR4 that has at least 90% sequence identity to FR1, FR2, FR3 and/or FR4 of the amino acid sequence set forth in SEQ ID NO. 40 or 62, respectively. In some embodiments, V L The region comprises the framework region 1 (FR 1), FR2, FR3 and/or FR4 sequence having at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence with the FR1, FR2, FR3 and/or FR4, respectively, of the amino acid sequence set forth in SEQ ID NO. 40 or 62Identity. In some embodiments, V L The regions comprise the framework region 1 (FR 1), FR2, FR3 and FR4 sequences that have at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to the FR1, FR2, FR3 and FR4, respectively, of the amino acid sequence set forth in SEQ ID NO. 40 or 62.
In some of any such embodiments, V L The region has the amino acid sequence set forth in SEQ ID NO. 40 or 62.
An anti-idiotype antibody or antigen-binding fragment thereof is provided comprising V set forth in SEQ ID NO. 36 or 58 H Amino acid sequences of CDR-H1, CDR-H2 and CDR-H3 sequences contained within the amino acid sequences of the regions; and/or amino acid sequences comprising CDR-L1, CDR-L2 and CDR-L3 sequences contained within the light chain Variable (VL) region amino acid sequences set forth in SEQ ID NO. 40 or 62.
Is provided with V H Region and V L Anti-idiotype antibodies or antigen-binding fragments thereof, of region V H The region has an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or% 99% identity to SEQ ID NO 36 or 58, said V L The region has an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID No. 40 or 62.
In some embodiments, there is provided a method comprising H Region and V L Anti-idiotype antibodies or antigen-binding fragments thereof, of region V H The region has the amino acid sequence set forth in SEQ ID NO 36 or 58, and V L The region has the amino acid sequence set forth in SEQ ID NO. 40 or 62. In some embodiments, antibodies are provided that contain the V set forth in SEQ ID NO. 36 H Region and V set forth in SEQ ID NO. 40 L A zone. In some embodiments, antibodies are provided that contain the V set forth in SEQ ID NO 58 H Region and V set forth in SEQ ID NO. 62 L A zone.
In some embodiments, the anti-idiotype antibody is a single chain antibody fragment, such as an scFv or diabody. In some implementationsIn embodiments, single chain antibodies include a linkage of two antibody domains or regions such as variable heavy chains (V H ) Region and variable light chain (V L ) Is provided for the connection of the one or more connectors. The linker is typically a peptide linker, such as a flexible and/or soluble peptide linker. Wherein the linker is a glycine and serine-rich and/or in some cases threonine-rich linker. In some embodiments, the linker further comprises charged residues, such as lysine and/or glutamic acid, which may increase solubility. In some embodiments, the linker further comprises one or more prolines.
In some embodiments, the anti-idiotype antibody is an intact antibody or a full length antibody. In some embodiments, the anti-ID may contain at least a portion of an immunoglobulin constant region, such as one or more constant region domains. In some embodiments, the constant regions comprise a light chain constant region (CL) and/or a heavy chain constant region 1 (CH 1). In some embodiments, the anti-ID comprises a CH2 and/or CH3 domain, such as an Fc region. In some embodiments, the Fc region is that of a human IgG (such as IgG1 or IgG 4). In some embodiments, the anti-idiotype antibody contains a CH domain set forth in SEQ ID NO. 37 or 59, or a portion thereof, or an amino acid sequence that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO. 37 or 59. In some embodiments, the anti-idiotype antibody contains a CL domain set forth in SEQ ID No. 41 or 63, or a portion thereof, or an amino acid sequence or portion thereof that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID No. 41 or 63.
In some embodiments, an anti-idiotype antibody specific for FMC63 comprises the heavy chain sequence set forth in SEQ ID NO 38 or 60 or a sequence exhibiting at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO 38 or 60 and/or comprises the light chain sequence set forth in SEQ ID NO 42 or 63 or a sequence exhibiting at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO 42 or 63. In some embodiments, an anti-idiotype antibody specific for FMC63 comprises the heavy chain sequence set forth in SEQ ID NO. 38 and/or the light chain sequence set forth in SEQ ID NO. 42. In some embodiments, an anti-idiotype antibody specific for FMC63 comprises the heavy chain sequence set forth in SEQ ID NO. 60 and/or the light chain sequence set forth in SEQ ID NO. 63. In some embodiments, the heavy and/or light chain of the anti-idiotype antibody further comprises a signal peptide. In some cases, the signal peptide has the sequence set forth in SEQ ID NO 39, 43, 61 or 64.
In some embodiments, the anti-idiotype antibody is an antigen binding fragment. In some embodiments, the antigen binding fragment is selected from the group consisting of: fragment antigen binding (Fab) fragments, F (ab') 2 Fragments, fab' fragments, fv fragments, single chain variable fragments (scFv), or single domain antibodies.
Thus, single chain antibody fragments, such as scFv and diabodies, particularly human single chain fragments, are provided which typically comprise a linkage of two anti-idiotype antibody domains or regions, such as V H And V L A linker of the domain. The linker is typically a peptide linker, e.g. a flexible and/or soluble peptide linker, such as a glycine and serine rich peptide linker.
In some aspects, the glycine and serine (and/or threonine) rich linker comprises at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of such amino acids. In some embodiments, they comprise at least or at least about 50%, 55%, 60%, 70%, or 75% glycine, serine, and/or threonine. In some embodiments, the linker consists essentially entirely of glycine, serine, and/or threonine. The linker length is typically between about 5 to about 50 amino acids, typically between 10 or about 10 to 30 or about 30, e.g., 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30, and in some examples, between 10 to 25 amino acids in length. Exemplary linkers include linkers having sequences GGGS (3 GS; SEQ ID NO: 29) or GGGGS (4 GS; SEQ ID NO: 26) with different numbers of repeats, such as 2, 3, 4, and 5 repeats of such sequences. Exemplary linkers include linkers having or consisting of the sequences set forth in SEQ ID NO. 25 (GGGGSGGGGSGGGGS). Exemplary linkers further include linkers having or consisting of the sequences set forth in SEQ ID NO. 33 (GSTSGSGKPGSGEGSTKG).
In some embodiments, the anti-idiotype antibody comprises an isolated antibody. In some embodiments, the anti-ID is humanized, recombinant, and/or monoclonal. In some embodiments, the anti-ID is a human anti-ID.
In some embodiments, the anti-idiotype antibody specific for FMC63 is humanized. In particular embodiments, all or substantially all CDR amino acid residues of a humanized anti-idiotype antibody specific for FMC63 are derived from non-human anti-FMC 63 CDRs. In some embodiments, the humanized anti-idiotype antibody specific for FMC63 comprises at least a portion of an antibody constant region derived from a human antibody.
In certain embodiments, the humanized anti-idiotype antibody specific for FMC63 comprises a human immunoglobulin (recipient antibody) in which residues from the heavy chain variable region of the recipient are replaced with residues from the heavy chain variable region of a non-human anti-idiotype antibody specific for FMC 63. In some cases, FR residues of the human immunoglobulin are replaced with corresponding non-human residues. In some embodiments, the humanized antibodies contain FR derived from a different gene. In some embodiments, the humanized anti-idiotype antibody specific for FMC63 comprises at least a portion of an immunoglobulin constant region (Fc), typically a constant region of a human immunoglobulin.
In some embodiments, the humanized anti-idiotype antibody specific for FMC63 contains an altered human acceptor antibody variable domain sequence that has been made to encode one or more amino acid residues corresponding to positions 4, 35, 38, 43, 44, 46, 58, 62, 64, 65, 66, 67, 68, 69, 73, 85, 98 (Kabat) and position 2, 4, 36, 39, 43, 45, 69, 70, 74, 75, 76, 78, 92 (Kabat) of the light chain variable region of the non-human donor sequence.
In certain embodiments, the anti-idiotype antibody specific for FMC63 is humanized. In specific embodiments, the humanized anti-idiotype antibody specific for FMC63 comprises one or more of CDR-L1, CDR-L2, CDR-L3 and CDR-H1, CDR-H2, CDR-H3 of a non-human anti-idiotype antibody specific for FMC 63. In some embodiments, some or all of the CDR-L1, CDR-L2, CDR-L3 and CDR-H1, CDR-H2, CDR-H3 regions contain one or more amino acid modifications. In some embodiments, the modification replaces a non-human amino acid residue with a human residue. In specific embodiments, one or more of CDR-L1, CDR-L2, CDR-L3 and CDR-H1, CDR-H2, CDR-H3 are inserted into the human FR region. In a specific embodiment, CDR-L1, CDR-L2, CDR-L3 and CDR-H1, CDR-H2, CDR-H3 of the non-human anti-idiotype antibody are CDRs of the VH region having the amino acid sequence set forth in SEQ ID NO 36 or 58. In certain embodiments, the CDRs of the non-human anti-idiotype VH region are or comprise CDR-H1 comprising the amino acid sequences set forth in SEQ ID NO 44, 65, 88, 89, 90, 98, 99 or 100; CDR-H2 comprising the amino acid sequence set forth in SEQ ID NO. 45, 66, 91, 92, 93, 101, 102 or 103; and/or CDR-H3 comprising the amino acid sequence set forth in SEQ ID NO 46, 67, 94 or 104. In some embodiments, the CDR-L1, CDR-L2, CDR-L3 and CDR-H1, CDR-H2, CDR-H3 of the non-human anti-idiotype antibody are CDRs of the VL region having the amino acid sequence set forth in SEQ ID NO. 40 or 62. In some embodiments, the CDRs of the non-human anti-idiotype VL region are or comprise CDR-L1 comprising the amino acid sequences set forth in SEQ ID NOS 47, 68, 95 or 105; CDR-L2 comprising the amino acid sequence set forth in SEQ ID NO 48, 69, 96 or 106; and/or CDR-L3 comprising the amino acid sequence set forth in SEQ ID NO 49, 97, 70 or 107. In some embodiments, all of the CDR-L1, CDR-L2, CDR-L3 and CDR-H1, CDR-H2, CDR-H3 regions of an anti-idiotype antibody specific for FMC63 are inserted into the FR of a human antibody. In specific embodiments, the CDR and FR regions are regions identified by Kabat, chothia, abM and/or Contact protocols.
In specific embodiments, one or more or all of CDR-L1, CDR-L2, CDR-L3 and CDR-H1, CDR-H2, CDR-H3 of a non-human anti-idiotype antibody specific for FMC63 is inserted into a framework region of a human antibody. In some embodiments, the human antibodies are IgA, igD, igE, igG and IgM antibodies. In particular embodiments, the human antibody is one of the subclasses of human IgA, igD, igE, igG and IgM, e.g., human IgG 1 、IgG 2 、IgG 3 、IgG 4 、IgA 1 Or IgA 2 . In some embodiments, one or more or all of CDR-L1, CDR-L2, CDR-L3 and CDR-H1, CDR-H2, CDR-H3 of a non-human anti-idiotype antibody specific for FMC63 is inserted into a framework region from and/or derived from an antigen binding region of a human antibody. In certain embodiments, the antigen binding fragment is from and/or derived from human IgA, igD, igE, igG and IgM antibodies. The subunit structure and three-dimensional configuration of different classes of human immunoglobulins are well known and are for example generally described by Abbas et al in Cellular and mol.immunology,4th ed. (w.b.samundrs, co., 2000). In some embodiments, the human antibody or antigen binding fragment thereof may be part of a larger fusion molecule formed by covalent or non-covalent binding of the human antibody to one or more other proteins or peptides.
In some embodiments, one or more or all of CDR-L1, CDR-L2, CDR-L3 and CDR-H1, CDR-H2, CDR-H3 of a non-human anti-idiotype antibody specific for FMC63 is inserted into a framework region of a human antibody or antigen binding fragment thereof that has all or part of an Fc region. In certain embodiments, the humanized anti-idiotype antibody specific for FMC63 contains all or part of the Fc region. In some embodiments, the Fc region has one or more modifications, such as amino acid modifications (e.g., substitutions, insertions, or deletions) at one or more amino acid positions. For example, such modifications may be made to improve half-life, alter binding to one or more types of Fc receptors, and/or alter effector function. In some embodiments, the modified Fc region alters (e.g., reduces) binding to the fcαr relative to binding of the unmodified Fc region to the fcαr. In certain embodiments, the humanized anti-idiotype antibody comprises all or a portion of a modified Fc region having altered (e.g., reduced) binding to an Fc receptor relative to the binding of an unmodified Fc region to an Fc receptor. Non-limiting examples of Fc modifications that alter binding to Fc receptors are described, for example, in U.S. patent nos. 7,217,797 and 7,732,570; and U.S. application nos. US 2010/0143254 and 2010/0143254.
In certain embodiments, humanized anti-idiotype antibodies specific for FMC63 have a heavy chain comprising the amino acid sequence set forth in SEQ ID NO. 138 and a light chain comprising the amino acid sequence set forth in SEQ ID NO. 139. In a specific embodiment, the humanized anti-idiotype antibody specific for FMC63 has a heavy chain comprising the amino acid sequence set forth in SEQ ID NO. 140 and a light chain comprising the amino acid sequence set forth in SEQ ID NO. 141. In some embodiments, humanized anti-idiotype antibodies specific for FMC63 have a heavy chain comprising the amino acid sequence set forth in SEQ ID NO:142 and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 143. In certain embodiments, humanized anti-idiotype antibodies specific for FMC63 have a heavy chain comprising the amino acid sequence set forth in SEQ ID NO. 144 and a light chain comprising the amino acid sequence set forth in SEQ ID NO. 145. In a specific embodiment, the humanized anti-idiotype antibody specific for FMC63 has a heavy chain comprising the amino acid sequence set forth in SEQ ID NO. 146 and a light chain comprising the amino acid sequence set forth in SEQ ID NO. 147. In some embodiments, humanized anti-idiotype antibodies specific for FMC63 have a heavy chain comprising the amino acid sequence set forth in SEQ ID NO. 148 and a light chain comprising the amino acid sequence set forth in SEQ ID NO. 149.
Exemplary features
The anti-idiotype antibodies provided herein may be identified, screened, or characterized by their physical/chemical properties and/or biological activity by a variety of known assays. In one aspect, the antigen binding activity of the anti-idiotype antibody is tested by known methods such as ELISA, western blotting and/or flow cytometry assays (including cell-based binding assays), e.g., the binding of the anti-idiotype antibody (e.g., conjugated or tagged with a fluorescent marker) to cells presenting the target anti-CD 19 antibody moiety is assessed and in some cases compared to the results using cells that do not express the target anti-CD 19 antibody moiety. Binding affinity can be measured as Kd or EC50.
In some embodiments of any of the antibodies provided, e.g., any of the antibodies provided in section a above, the target anti-CD 19 antibody moiety is SJ25C1 or an antibody derived from SJ25C 1.
In some embodiments of any of the antibodies provided, e.g., any of the antibodies provided in section B above, the target anti-CD 19 antibody moiety is FMC63 or an antibody derived from FMC 63.
Competition assays can be used to identify antibodies that compete with any of the anti-idiotype antibodies described herein. Assays for mapping epitopes bound by anti-idiotype antibodies and reference antibodies can also be used and are known.
In some embodiments, the anti-idiotype antibody does not cross-react with an anti-CD 19 antibody moiety other than the target anti-CD 19 antibody moiety. In some embodiments, the target anti-CD 19 antibody moiety is derived from an SJ25C1 antibody. In some embodiments, the target anti-CD 19 antibody moiety is derived from the SJ25C1 antibody and the anti-idiotype antibody does not cross-react with an anti-CD 19 antibody moiety derived from the FMC63 antibody, said anti-CD 19 antibody comprising V comprising the amino acid sequence of SEQ ID No. 30 H And V comprising the amino acid sequence SEQ ID NO. 31 L . In some embodiments, the target anti-CD 19 antibody moiety is derived from an FMC63 antibody. In some embodiments, the target anti-CD 19 antibody moiety is derived from an FMC63 antibody and the anti-idiotype antibody does not cross-react with an anti-CD 19 antibody moiety derived from an SJ25C1 antibody, the anti-CD 19 antibody comprising a V comprising the amino acid sequence SEQ ID NO:23 H And V comprising the amino acid sequence SEQ ID NO. 24 L 。
In some embodiments, the anti-idiotype antibody specifically binds to a target anti-CD 19 antibody moiety that is part of a fusion protein such as a recombinant receptor. In some embodiments, the anti-idiotype antibody does not bind to any epitope in the fusion protein outside the target anti-CD 19 antibody moiety. For example, in some embodiments, the target anti-CD 19 antibody moiety is or is part of the antigen binding domain of a Chimeric Antigen Receptor (CAR), and the anti-idiotype antibody does not bind any epitope outside of the antigen binding domain. In some embodiments, the CAR antigen binding domain comprises or consists of an scFv.
In some embodiments, the anti-idiotype antibody specifically binds to a target anti-CD 19 antibody moiety that is an scFv contained in the CAR. In some embodiments, the anti-idiotype antibody specifically binds to overlapping epitopes of one or more Complementarity Determining Regions (CDRs) of the target anti-CD 19 scFv. In some embodiments, the anti-idiotype antibody does not bind any epitope in the CAR outside the scFv; in some embodiments, it does not bind to a reference antibody. In some embodiments, the reference antibody and the target antibody specifically bind to the same antigen, e.g., they bind to CD19 and/or comprise one or more variable heavy and/or variable light chain framework regions having at least 90%, 95%, 96%, 97%, 98% or 99% identity to the corresponding framework regions of the target antibody (in some aspects, the one or more framework regions comprise FR1, FR2, FR3 and/or FR4 of the heavy and/or light chain); and/or contain (or use) the same heavy and/or light chain v gene as the target antibody and/or are derived from the same v gene sequence as the target antibody. In some aspects, the reference antibody is FMC63. In some aspects, the reference antibody is SJ25C1. In some embodiments, the CAR comprises a spacer that links the scFv to its transmembrane domain, and the anti-idiotype antibody does not bind any epitope in the spacer. In some embodiments, the spacer is a sequence derived from CD28, such as an extracellular portion from CD 28. In some embodiments, the spacer comprises the amino acid sequence SEQ ID NO 27. In some embodiments, the anti-idiotype antibody does not bind any epitope in an Fc domain (such as the Fc domain of IgG 1). In some embodiments, the Fc domain is an IgG1Fc domain lacking a hinge region. In some embodiments, the Fc domain comprises the amino acid sequence SEQ ID NO. 32.
In some embodiments, the anti-idiotype antibody does not cross-react with a different CAR. In some embodiments, the anti-idiotype antibody does not cross-react with a different anti-CD 19 CAR. In some embodiments, the anti-idiotype antibody does not cross-react with an anti-CD 19 antibody moiety (e.g., of a reference antibody) that has one or more distinct unique positions compared to the target anti-CD 19 scFv. In some embodiments, the anti-idiotype antibody is specific for a target anti-CD 19scFv of a CAR derived from the SJ25C1 antibody. In some embodiments, the target anti-CD 19 antibody moiety is derived from the SJ25C1 antibody, and the anti-idiotype antibody does not cross-react with a CAR containing an anti-CD 19 antibody moiety derived from the FMC63 antibody. In some embodiments, the anti-idiotype antibody is specific for a target anti-CD 19scFv of a CAR derived from an FMC63 antibody. In some embodiments, the target anti-CD 19 antibody moiety is derived from an FMC63 antibody, and the anti-idiotype antibody does not cross-react with a CAR containing an anti-CD 19 antibody moiety derived from an SJ25C1 antibody.
In some embodiments, the anti-idiotype antibody is an agonist of the CAR. In some embodiments, the anti-idiotype antibody is an antagonist of a CAR.
In some embodiments, the provided anti-idiotype antibodies are capable of binding a target anti-CD 19 module (such as antibody SJ25C1 or FMC 63) with at least some affinity, as measured by any of a number of known methods. In some embodiments, affinity is such as to balance dissociation constants (K D ) A representation; in some embodiments, affinity is expressed as EC 50 And (3) representing. In certain embodiments, the anti-idiotype antibody has a binding affinity (EC 50) and/or dissociation constant for the anti-CD 19 module that is equal to or about (or less than about) 100nM, 50nM, 40nM, 30nM, 25nM, 20nM, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1nM, such as between equal to or about 1nM and equal to or about 15nM, for example, between equal to or about 5nM and equal to or about 10 nM. In one embodiment, the degree of binding of the anti-idiotype antibody to a moiety unrelated to the target anti-CD 19 moiety is less than 10% or about 10% of the binding of the antibody to the target anti-CD 19 moiety, as for example by radiationAs measured by immunoassay (RIA).
Nucleic acid
Nucleic acids encoding antibodies and/or portions thereof (e.g., strands thereof) are also provided. Wherein the nucleic acid provided is a nucleic acid encoding an anti-idiotype antibody described herein. Nucleic acids may include nucleic acids comprising naturally and/or non-naturally occurring nucleotides and bases, including for example nucleic acids having backbone modifications. The terms "nucleic acid molecule", "nucleic acid" and "polynucleotide" are used interchangeably and refer to a polymer of nucleotides. Such nucleotide polymers may contain natural and/or unnatural nucleotides, and include, but are not limited to, DNA, RNA, and PNA. "nucleic acid sequence" refers to a linear sequence of nucleotides, including nucleic acid molecules or polynucleotides. Exemplary nucleic acids and vectors are those having the sequences set forth in SEQ ID NOS 15-22, 50-57, 71-77 and CDR encoding portions thereof, as well as nucleic acids and vectors containing sequences having at least or at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the sequences. The nucleic acid may encode a V comprising an anti-idiotype antibody L And/or V comprising an anti-idiotype antibody H For example, the light and/or heavy chain of the antibody.
Vectors containing the nucleic acids, host cells containing the vectors, e.g., for producing the antibodies, are also provided. Methods for producing the antibodies are also provided. In a further embodiment, one or more vectors (e.g., expression vectors) comprising such nucleic acids are provided. In a further embodiment, a host cell comprising such a nucleic acid is provided. In one such embodiment, the host cell comprises (e.g., has been transformed with): (1) Comprising encoding a polypeptide comprising antibody V L Amino acid sequence of (c) and comprising antibody V H A vector comprising a nucleic acid encoding an amino acid sequence comprising antibody V, or (2) a nucleic acid comprising an amino acid sequence comprising an antibody V L First vector comprising a nucleic acid encoding an amino acid sequence comprising antibody V H A second vector of a nucleic acid of an amino acid sequence of (a). In some embodiments, methods of making an anti-idiotype antibody are provided, wherein the methods comprise, where appropriateCulturing a host cell comprising a nucleic acid encoding the antibody as provided above under conditions for expression of the antibody, and optionally recovering the antibody from the host cell (or host cell culture medium).
Method for producing antibodies
Methods of making an anti-idiotype antibody of any of the provided embodiments are also provided. In some embodiments, for recombinant production of an anti-idiotype antibody, a nucleic acid encoding an antibody, e.g., as described above, may be isolated and inserted into one or more vectors for further cloning and/or expression in a host cell. Such nucleic acids can be readily isolated and sequenced using conventional procedures, such as by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of the antibody.
In addition to prokaryotes, eukaryotic microorganisms such as filamentous fungi or yeast are suitable cloning or expression hosts for vectors encoding antibodies, including fungal and yeast strains whose glycosylation pathways have been modified to mimic or approximate those in human cells resulting in the production of antibodies with a partially or fully human glycosylation pattern. See Gerngross, nat.Biotech.22:1409-1414 (2004), and Li et al, nat.Biotech.24:210-215 (2006).
Exemplary eukaryotic cells that can be used to express the polypeptide include, but are not limited to, COS cells, including COS 7 cells; 293 cells, including 293-6E cells; CHO cells, including CHO-S, dg44.lec13cho cells and FUT8CHO cells; A cell; and NSO cells. In some embodiments, antibody heavy and/or light chains may be expressed in yeast. See, for example, U.S. publication No. US 2006/0270045 A1. In some embodiments, the selection of a particular eukaryotic host cell is based on its ability to make a desired post-translational modification of the heavy and/or light chain. For example, in some embodiments, CHO cells produce a polypeptide having a higher level of sialylation than the same polypeptide produced in 293 cells.
In some embodiments, the anti-idiotype antibody is produced in a cell-free system. Exemplary cell-free systems are described, for example, in Sitaraman et al, methods mol. Biol.498:229-44 (2009); spirin, trends Biotechnol.22:538-45 (2004); endo et al, biotechnol. Adv.21:695-713 (2003).
The embodiments provided further include vectors and host cells for expression and production of antibodies and other binding proteins, as well as other expression systems, including eukaryotic and prokaryotic host cells, including bacteria, filamentous fungi, and yeast, as well as mammalian cells (e.g., human cells), and cell-free expression systems.
The anti-idiotype antibody or antibody moiety may be a humanized antibody or a human antibody. A "humanized" antibody is one in which all or substantially all CDR amino acid residues are derived from non-human CDRs and all or substantially all FR amino acid residues are derived from human FRs. The humanized antibody optionally may comprise at least a portion of an antibody constant region derived from a human antibody. "humanized form" of a non-human antibody refers to a variant of a non-human antibody that has undergone humanization, typically in order to reduce immunogenicity to humans, while retaining the specificity and affinity of the parent non-human antibody. In some embodiments, some FR residues in a humanized antibody are substituted with corresponding residues from a non-human antibody (e.g., an antibody from which CDR residues are derived), e.g., for restoring or increasing antibody specificity or affinity.
Wherein the anti-idiotype antibody or antibody moiety provided is a human antibody. A "human antibody" is an antibody having an amino acid sequence that corresponds to an amino acid sequence of an antibody produced by a human or human cell, or an antibody produced by a non-human source (including a human antibody library) using a human antibody repertoire or other human antibody coding sequence. The term excludes humanized versions of non-human antibodies that comprise non-human antigen binding regions, such as those antibodies in which all or substantially all of the CDRs are non-human.
Human antibodies can be prepared by administering an immunogen to a transgenic animal that has been modified to produce whole human antibodies or whole antibodies having human variable regions responsive to antigen challenge. Such animals typically contain all or part of the human immunoglobulin loci, either in place of endogenous immunoglobulin loci, or extrachromosomally present or randomly integrated into the animal chromosome. In such transgenic animals, the endogenous immunoglobulin loci have typically been inactivated. Human antibodies can also be derived from human antibody libraries, including phage display libraries and cell-free libraries, which contain antibody coding sequences derived from human repertoires.
Immunoconjugates
In some embodiments, the anti-idiotype antibody is an immunoconjugate (anti-idiotype antibody immunoconjugate) or portion thereof, wherein the anti-idiotype antibody is conjugated to one or more heterologous molecules (such as, but not limited to, a cytotoxic agent or imaging agent). Cytotoxic agents include, but are not limited to, radioisotopes (e.g., radioisotopes of At211, I131, I125, Y90, re186, re188, sm153, bi212, P32, pb212, and Lu); chemotherapeutic agents (e.g., maytansinoids, taxanes, methotrexate, doxorubicin, vinca alkaloids (vincristine, vinblastine, etoposide), doxorubicin, melphalan, mitomycin C, chlorambucil, daunorubicin, or other intercalating agents); a growth inhibitor; enzymes and fragments thereof, such as nucleolytic enzymes; an antibiotic; toxins, such as small molecule toxins or enzymatically active toxins. In some embodiments, the antibody is conjugated to one or more cytotoxic agents such as chemotherapeutic agents or drugs, growth inhibitory agents, toxins (e.g., bacterial, fungal, plant or animal derived protein toxins, enzymatically active toxins, or fragments thereof), or radioisotopes.
Wherein the anti-idiotype antibody immunoconjugate is an antibody-drug conjugate (ADC), wherein the idiotype antibody is conjugated to one or more drugs, including but not limited to: maytansinoids (see U.S. Pat. nos. 5,208,020, 5,416,064 and european patent EP 0 425 235b 1); auristatins, such as monomethyl auristatin drug modules DE and DF (MMAE and MMAF) (see U.S. Pat. nos. 5,635,483 and 5,780,588, and 7,498,298); dolastatin; calicheamicin or derivatives thereof (see U.S. Pat. Nos. 5,712,374, 5,714,586, 5,739,116, 5,767,285, 5,770,701, 5,770,710, 5,773,001, and 5,877,296; hinman et al, cancer Res.53:3336-3342 (1993), and Lode et al, cancer Res.58:2925-2928 (1998)); anthracyclines such as daunorubicin or doxorubicin (see Kratz et al, current Med. Chem.13:477-523 (2006), jeffrey et al, bioorganic & Med. Chem. Letters 16:358-362 (2006), torgov et al, bioconj. Chem.16:717-721 (2005), nagy et al, proc. Natl. Acad. Sci. USA97:829-834 (2000), dubowchik et al, bioorg et al, med. Chem. Letters 12:1529-1532 (2002), king et al, J. Med. Chem.45:4336-4343 (2002), and U.S. Pat. No.6,630,579); methotrexate; vindesine; taxanes, such as docetaxel, paclitaxel, ralostazol, tesetaxel, and ostazol; trichothecene; and CC1065.
And wherein the anti-idiotype antibody immunoconjugate is a conjugate in which the antibody is conjugated to an enzymatically active toxin or fragment thereof, including but not limited to: diphtheria chain, non-binding active fragments of diphtheria toxin, exotoxin a chain (from pseudomonas aeruginosa), ricin a chain, abrin a chain, curdlan a chain, alpha-curcin, aleurone, caryophyllin protein, pokeweed protein (PAPI, PAPII and PAP-S), balsam pear inhibitors, curcin, crotylosin, soapbark inhibitors, gelonin, mi Tuojun, curcin, phenomycin, ionomycin and trichothecene toxoid.
And wherein the anti-idiotype antibody immunoconjugates are those wherein the anti-idiotype antibody is conjugated to a radioactive atom to form a radioactive conjugate. Exemplary radioisotopes include At 211 、I 131 、I 125 、Y 90 、Re 186 、Re 188 、Sm 153 、Bi 212 、P 32 、Pb 212 And a radioisotope of Lu.
Conjugates of anti-idiotype antibodies and cytotoxic agents can be made using any of a number of known protein coupling agents, such as linkers (see Vitetta et al, science 238:1098 (1987)), WO94/11026. The linker may be a "cleavable linker" that facilitates release of the cytotoxic drug in the cell, such as an acid labile linker, a peptidase sensitive linker, a photolabile linker, a dimethyl linker, and a disulfide-containing linker (Chari et al, cancer Res.52:127-131 (1992); U.S. Pat. No.5,208,020).
Also provided are anti-idiotype antibody immunoconjugates comprising an anti-idiotype antibody attached to a label, such as a detectable label, that can indirectly or directly produce a detectable signal. These anti-idiotype antibody immunoconjugates can be used in research or diagnostic applications. The label is preferably capable of producing a detectable signal, either directly or indirectly. For example, the marker may be a radio-opaque or radioisotope, such as 3 H、 14 C、 32 P、 35 S、 123 I、 125 I、 131 I, a step of I; fluorescent (fluorophore) or chemiluminescent (chromophore) compounds, such as fluorescein isothiocyanate, rhodamine or luciferin; enzymes such as alkaline phosphatase, beta-galactosidase, or horseradish peroxidase; an imaging agent; or metal ions. In some embodiments, the label is a radioactive atom for scintigraphy studies, e.g 99 Tc or 123 I, or spin labeling for Nuclear Magnetic Resonance (NMR) imaging (also known as magnetic resonance imaging, MRI), such as zirconium-89, iodine-123, iodine-131, indium-111, fluorine-19, carbon-13, nitrogen-15, oxygen-17, gadolinium, manganese, or iron. Zirconium-89 can be complexed with various metal chelators and conjugated to antibodies, for example for PET imaging (WO 2011/056983).
Examples of detectable labels include, but are not limited to, radionuclides (radionuclides), enzymes, coenzymes, fluorescers, chemiluminescent agents (chemiluminescers), chromogens, enzyme substrates or cofactors, enzyme inhibitors, cofactor complexes, free radicals, particles, dyes, and the like. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, beta-galactosidase, or acetylcholinesterase; examples of suitable prosthetic groups include streptavidin/biotin and avidin/biotin; examples of suitable fluorescent materials include umbelliferone, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein (dichlorotriazinylamine fluorescein), dansyl chloride or phycoerythrin, coumarin, alexa488, oregon green 488, rhodamine green, alexa 532, cy3, bodipy 588/586, alexa586, TAMRA, rox, alexa 594, texas Red (Texas Red), bodipy 630/650, cy5, alexa647, IR dye 680, IR dye 700DX, cy5.5, alexa750, IR dye 800CW, IR dye 800, atto 532, and Atto 465.
In some embodiments, the anti-idiotype antibody immunoconjugate is directly detectable. For example, a secondary antibody specific for the anti-idiotype antibody immunoconjugate and containing a detectable label may be used to detect the anti-idiotype antibody immunoconjugate.
Multispecific antibodies
In certain embodiments, the anti-idiotype antibody is multispecific. Wherein the multispecific binding molecule is a multispecific antibody, including, for example, a bispecific antibody. A multispecific binding partner (e.g., an antibody) has binding specificity for at least two different sites, which may be in the same or different antigens. In certain embodiments, one of the binding specificities is for an anti-CD 19 antibody moiety, while the other is for another antigen. In certain embodiments, the bispecific antibody may bind to two different epitopes of an anti-CD 19 antibody moiety. Bispecific antibodies can also be used to localize cytotoxic agents to cells expressing an anti-CD 19 antibody moiety on their surface, such as anti-CD 19CAR T cells. Bispecific antibodies can be prepared in the form of full length antibodies or antibody fragments. Wherein the bispecific antibody is a specific single chain antibody, such as diabodies, triabodies and tetrabodies, tandem di-scFv and tandem tri-scFv.
Variants
In certain embodiments, an anti-idiotype antibody comprises one or more amino acid variations, such as substitutions, deletions, insertions, and/or mutations, as compared to the sequences of the anti-idiotype antibodies described herein. Exemplary variants include variants designed to improve the binding affinity and/or other biological properties of an anti-idiotype antibody. Amino acid sequence variants of anti-idiotype antibodies can be prepared by introducing appropriate modifications into the nucleotide sequence encoding the anti-idiotype antibody or by peptide synthesis. Such modifications include, for example, deletions and/or insertions and/or substitutions of residues in the amino acid sequence of the anti-idiotype antibody. Any combination of deletions, insertions, and substitutions may be made to arrive at the final construct, provided that the final construct has the desired characteristics, such as antigen binding.
In certain embodiments, for example, an anti-idiotype antibody comprises one or more amino acid substitutions as compared to the anti-idiotype antibody sequences described herein. Sites of interest for substitution mutagenesis include CDRs and FR. Amino acid substitutions may be introduced into an anti-idiotype antibody of interest and the product screened for desired activity, e.g., retention/improved antigen binding, reduced immunogenicity, improved half-life, and/or improved effector functions, such as the ability to promote antibody-dependent cell-mediated cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC). In some embodiments, the variant anti-idiotype antibody exhibits retained or improved binding to the target anti-CD 19 antibody or fragment thereof. For example, in some embodiments, the variant anti-idiotype antibody exhibits an increase in binding affinity for the target anti-CD 19 antibody of at least about 10% (such as at least about any one of 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, 1000% or more) compared to the unmodified anti-idiotype antibody.
In some embodiments, one or more residues within the CDRs of a parent antibody (e.g., a humanized antibody or a human antibody) are substituted. In some embodiments, substitutions are made to restore the sequence or position in the sequence to a germline sequence, such as an antibody sequence found in a germline (e.g., human germline), in order to reduce the likelihood of immunogenicity, e.g., when administered to a human individual.
In some embodiments, changes may be made in CDR "hot spots," i.e., residues encoded by codons that mutate at high frequencies during somatic maturation (see, e.g., chordhury, methods mol. Biol.207:179-196 (2008)), and/or graftingResidues of haptens, wherein the resulting variants V were tested H Or V L Is used for the binding affinity of (a) to the substrate. Affinity maturation by construction and reselection from secondary libraries has been described, for example, in Hoogenboom et al, from Methods in Molecular Biology 178:178:1-37 (edited by O' Brien et al, human Press, totowa, N.J. (2001)). In some embodiments of affinity maturation, diversity is introduced into the variable gene selected for maturation by any of a variety of methods (e.g., error-prone PCR, strand shuffling, or oligonucleotide-directed mutagenesis). A secondary library is then created. The library is then screened to identify any antibody variants with the desired affinity. Another approach to introducing diversity involves CDR-directed approaches in which several CDR residues (e.g., 4-6 residues at a time) are randomized. CDR residues involved in antigen binding can be specifically identified, for example, using alanine scanning mutagenesis or modeling. In particular, CDR-H3 and CDR-L3 are often targeted.
In certain embodiments, substitutions, insertions, or deletions may be made within one or more CDRs, provided that such changes do not substantially reduce the ability of the antibody to bind to an antigen. For example, conservative changes (e.g., conservative substitutions as provided herein) may be made in the CDRs that do not substantially reduce binding affinity. Such alterations may be, for example, outside of the antigen-contacting residues in the CDRs. Variant V provided above H And V L In certain embodiments of the sequence, each CDR is unchanged or contains no more than one, two, or three amino acid substitutions.
Amino acid sequence insertions include amino and/or carboxy terminal fusions ranging in length from one residue to polypeptides containing one hundred or more residues, as well as intersystem insertions of single or multiple amino acid residues. Examples of terminal insertions include antibodies with an N-terminal methionyl residue. Other insertional variants of an antibody molecule include fusions of the N-or C-terminus of the antibody with an enzyme or a polypeptide that increases the serum half-life of the antibody.
Modification
In certain embodiments, for example, certain cell lines are used, e.g., by altering antibodies by removing or inserting one or more glycosylation sites via altering the amino acid sequence and/or by modifying oligosaccharides attached to the glycosylation sites, to increase or decrease the degree of antibody glycosylation.
Exemplary modifications, variants and cell lines are described, for example, in patent publication nos. US 2003/0157108, US 2004/0093621, US 2003/0157108; WO 2000/61739; WO 2001/29246; US 2003/015614; US 2002/0164328; US 2004/0093621; US 2004/013321; US 2004/010704; US 2004/0110282; US 2004/0109865; WO 2003/085119; WO 2003/084570; WO 2005/035586; WO 2005/035778; WO2005/053742; WO2002/031140; okazaki et al, J.mol.biol.336:1239-1249 (2004); yamane-Ohnuki et al, biotech.bioeng.87:614 (2004), ripka et al, arch.biochem.Biophys.249:533-545 (1986); U.S. patent application Ser. No. 2003/0157108 A1,Presta,L; and WO 2004/056312A1, yamane-Ohnuki et al, biotech. Bioeng.87:614 (2004); kanda, y et al, biotechnol. Bioeng.,94 (4): 680-688 (2006); and WO 2003/085107); WO 2003/01878 (Jean-Maiset et al); U.S. Pat. No.6,602,684 (Umana et al); and US 2005/0123946 (Umana et al); WO 1997/30087 (Patel et al); WO 1998/58964 (Raju, s.); and WO 1999/22764 (Raju, S.).
Wherein the modified antibodies are antibodies having one or more amino acid modifications in the Fc region, such as those having a human Fc region sequence or other portion of the constant region (e.g., a human IgG1, igG2, igG3, or IgG4Fc region) comprising amino acid modifications (e.g., substitutions) at one or more amino acid positions.
For example, such modifications may be made to improve half-life, alter binding to one or more types of Fc receptors, and/or alter effector function.
And wherein the variants are cysteine engineered antibodies, such as "thioMAbs" and other cysteine engineered variants, wherein one or more residues of the antibody are substituted with cysteine residues to generate reactive thiol groups at accessible sites, such as for conjugation of agents and linkers, to produce immunoconjugates. Cysteine engineered antibodies are described, for example, in U.S. Pat. nos. 7,855,275 and 7,521,541.
In some embodiments, the antibodies are modified to contain additional non-protein moieties, including water-soluble polymers. Exemplary polymers include, but are not limited to, polyethylene glycol (PEG), ethylene glycol/propylene glycol copolymers, carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, poly-1, 3-dioxolane, poly-1, 3, 6-trioxane, ethylene/maleic anhydride copolymers, polyaminoacids (homo-or random copolymers) and dextran or poly (n-vinylpyrrolidone) polyethylene glycol, propylene glycol homopolymers, polyoxypropylene/ethylene oxide copolymers, polyoxyethylated polyols (e.g., glycerin), polyvinyl alcohol, and mixtures thereof. Polyethylene glycol propionaldehyde has manufacturing advantages due to its stability in water. The polymer may be of any molecular weight and may be branched or unbranched. The number of polymers attached to the antibody may vary, and if more than one polymer is attached, they may be the same or different molecules. In general, the number and/or type of polymers used for derivatization may be determined based on considerations including, but not limited to, the particular nature or function of the antibody to be improved, whether the antibody derivative is to be used in therapy under defined conditions, and the like.
Method for identifying anti-idiotype antibodies
In some embodiments, methods are provided for identifying anti-idiotype antibodies or antigen-binding fragments that specifically bind to a target antibody or antigen-binding fragment thereof, such as by using a hybridoma method with an immunogen comprising the target antibody or fragment thereof (see, e.g., kohler and Milstein, nature,256:495 (1975) and Sergeeva et al, blood,117 (16): 4262-4272). The immunogen may comprise an immunogenicity enhancing moiety fused to the target antibody or fragment. Such an immunogenicity enhancing module may have a variety of characteristics including, but not limited to, increasing the solubility and half-life of the immunogen. Exemplary immunogenicity enhancing modules include Fc domains or fragments thereof. In some embodiments, the immunogenicity enhancing module is an Fc domain (such as optionally from human IgG 1). In some embodiments, the immunogenicity enhancing module is an Fc domain lacking all or part of the hinge region (such as optionally from human IgG 1).
In some embodiments, methods are provided for identifying an anti-idiotype antibody or antigen-binding fragment that specifically binds to a target antibody or antigen-binding fragment thereof, the method comprising: (a) Introducing a soluble immunogen comprising an antigen binding fragment of a target antibody fused to an immunogenicity enhancing moiety to a subject; and (b) identifying an antibody from the subject that specifically binds to the target antibody or antigen-binding fragment thereof. In some embodiments, the antigen binding fragment comprises a variable heavy chain region and/or a variable light chain region of the target antibody. In some embodiments, the antigen binding fragment is a single-stranded fragment. In some embodiments, the antigen binding fragment is an scFv. In some embodiments, the antigen binding fragment is within or contained within an antigen binding domain of the extracellular portion of a Chimeric Antigen Receptor (CAR).
In some embodiments, the immunogenicity enhancing module is an Fc domain or fragment thereof, optionally a human IgG1Fc. In some embodiments, the immunogenicity enhancing module is an Fc domain lacking a hinge region. In some embodiments, the immunogenicity enhancing module comprises the amino acid sequence set forth in SEQ ID NO. 32.
In some embodiments, identifying antibodies comprises identifying individual antibody clones produced by a subject using hybridoma technology, and screening the clones for binding to a target antibody or antigen-binding fragment thereof. In some embodiments, identifying the antibody comprises: (i) Isolating B cells from the spleen of the subject and fusing them with immortalized B cells to produce hybridomas; (ii) Screening the hybridomas for the production of antibodies that specifically bind to the target antibody or antigen-binding fragment thereof or chimeric antigen receptor comprising the antigen-binding fragment; and (iii) sequencing the antibodies from the hybridoma producing the antibody that specifically binds to the target antibody or antigen binding fragment, thereby identifying the anti-idiotype antibody. In some embodiments, screening hybridomas includes determining the binding affinity of the hybridoma antibody to a target molecule, such as an scFv or a CAR or fragment thereof, comprising the heavy and/or light chain variable regions or portions thereof (such as one or more CDRs of the VH and/or VL regions) of the target antibody or a unique position of the target antibody. In some embodiments, screening hybridomas further comprises determining the binding affinity of a hybridoma antibody to a non-target molecule that does not comprise a unique position of the target antibody, such as Fc or a fragment thereof, or another antibody or fragment thereof that does not comprise the heavy and/or light chain variable regions of the target antibody, or portions thereof (such as one or more CDRs of the VH and/or VL regions), wherein binding of the hybridoma antibody to the target molecule but not to the non-target molecule indicates that the hybridoma antibody specifically binds the target antibody.
In some of any such embodiments, the method comprises: (a) Introducing a soluble immunogen comprising an scFv of a target antibody fused to an Fc domain or fragment thereof to a subject; and (b) identifying antibodies from the subject that specifically bind to a molecule (such as an immunogen) comprising a unique site of the target antibody. In some embodiments, the scFv is within or contained within an antigen binding domain of an extracellular portion of a Chimeric Antigen Receptor (CAR). In some embodiments, the Fc domain or fragment thereof is a human IgG1Fc or fragment thereof. In some embodiments, the Fc domain or fragment thereof is an Fc domain lacking a hinge region. In some embodiments, the Fc domain or fragment thereof comprises the amino acid sequence set forth in SEQ ID NO. 32. In some embodiments, identifying antibodies includes identifying individual antibody clones produced by a subject using hybridoma technology, and screening the antibody clones for binding to a molecule (such as an immunogen) comprising a unique site of a target antibody. In some embodiments, identifying the antibody comprises: (i) Isolating B cells from the spleen of the subject and fusing them with immortalized B cells to produce hybridomas; (ii) Screening hybridomas for the production of antibodies that specifically bind to a molecule (e.g., an immunogen) comprising a unique position of a target antibody; and (iii) sequencing antibodies from hybridomas producing antibodies that specifically bind to molecules (e.g., immunogens) comprising the unique positions of the target antibody, thereby identifying anti-idiotype antibodies. In some embodiments, screening hybridomas includes determining the binding affinity of the hybridoma antibody to a target molecule comprising a unique position of the target antibody, such as an immunogen, an scFv, or a CAR or fragment thereof comprising the heavy and/or light chain variable regions of the target antibody or portions thereof (such as one or more CDRs of the VH and/or VL regions). In some embodiments, screening hybridomas further comprises determining the binding affinity of a hybridoma antibody to a non-target molecule that does not comprise a unique position of the target antibody, such as Fc or a fragment thereof, or another antibody or fragment thereof that does not comprise the heavy and/or light chain variable regions of the target antibody, or portions thereof (such as one or more CDRs of the VH and/or VL regions), wherein binding of the hybridoma antibody to the target molecule but not to the non-target molecule indicates that the hybridoma antibody specifically binds the target antibody.
In some of any such embodiments, the method comprises: (a) Introducing a soluble immunogen comprising an scFv of a target antibody fused to an Fc domain or fragment thereof to a subject; and (b) identifying an antibody from the subject that (i) binds to a target molecule comprising a unique position of the target antibody, such as an immunogen, an scFv, or a CAR or fragment thereof comprising the heavy and/or light chain variable regions of the target antibody or portions thereof (such as one or more CDRs of the VH and/or VL regions); (ii) A non-target molecule such as Fc or a fragment thereof or another antibody or fragment thereof that does not comprise the heavy and/or light chain variable regions or portions thereof (such as one or more CDRs of the VH and/or VL regions) of the target antibody. In some embodiments, the scFv is within or contained within an antigen binding domain of an extracellular portion of a Chimeric Antigen Receptor (CAR). In some embodiments, the Fc domain or fragment thereof is a human IgG1Fc or fragment thereof. In some embodiments, the Fc domain or fragment thereof is an Fc domain lacking a hinge region. In some embodiments, the Fc domain or fragment thereof comprises the amino acid sequence set forth in SEQ ID NO. 32. In some embodiments, identifying antibodies includes identifying individual antibody clones produced by a subject using hybridoma technology, and screening the antibody clones for binding to target molecules and non-target molecules. In some embodiments, identifying the antibody comprises: (i) Isolating B cells from the spleen of the subject and fusing them with immortalized B cells to produce hybridomas; (ii) Screening hybridomas for the production of antibodies which bind to target molecules but not to non-target molecules; and (iii) sequencing the antibodies from the hybridoma producing antibodies that bind to the target molecule but not to the non-target molecule, thereby identifying the anti-idiotype antibody. In some embodiments, screening hybridomas includes determining binding affinity of the hybridoma antibodies to target molecules and non-target molecules.
In some of any such embodiments, the method comprises: (a) Introducing into a subject a soluble immunogen comprising an scFv derived from an FMC63 antibody (such as an scFv comprising the amino acid sequence SEQ ID NO: 34) fused to an Fc domain or fragment thereof; and (b) identifying an antibody from the subject that (i) binds to a target molecule comprising a unique position of an FMC63 antibody, such as an immunogen, scFv, or CAR or fragment thereof comprising the heavy and/or light chain variable region of an FMC63 antibody or a portion thereof (such as one or more CDRs of the VH and/or VL regions); (ii) Non-target molecules such as Fc or fragments thereof or another antibody or fragment thereof that does not comprise the heavy and/or light chain variable regions of FMC63 antibodies or portions thereof (such as one or more CDRs of VH and/or VL regions) that do not comprise the unique positions of FMC63 antibodies. In some embodiments, the immunogen comprises the amino acid sequences set forth in SEQ ID NO. 34 and SEQ ID NO. 32, optionally separated by a linker (e.g., set forth in SEQ ID NO. 33). In some embodiments, the immunogen comprises the amino acid sequence SEQ ID NO. 35. In some embodiments, the antibody identified in (b) binds to an immunogen and does not bind to an Fc domain or fragment thereof or a molecule comprising an scFv derived from an SJ25C1 antibody (such as an scFv comprising the amino acid sequence SEQ ID NO: 28). In some embodiments, the antibody identified in (b) binds to a target molecule comprising amino acid sequence SEQ ID NO. 34 or 35 and does not bind to a non-target molecule comprising amino acid sequence SEQ ID NO. 32 or a non-target molecule comprising amino acid sequence SEQ ID NO. 28.
In some of any such embodiments, the method comprises: (a) Introducing into a subject a soluble immunogen comprising an scFv derived from an SJ25C1 antibody (such as an scFv comprising the amino acid sequence SEQ ID NO: 28) fused to an Fc domain or fragment thereof; and (b) identifying an antibody from the subject that (i) binds to a target molecule comprising a unique position of the SJ25C1 antibody, such as an immunogen, scFv, or CAR or fragment thereof comprising the heavy and/or light chain variable regions or portions thereof (such as one or more CDRs of the VH and/or VL regions) of the SJ25C1 antibody; (ii) A non-target molecule such as Fc or a fragment thereof or another antibody or fragment thereof that does not comprise the heavy and/or light chain variable regions of an SJ25C1 antibody or portions thereof (such as one or more CDRs of the VH and/or VL regions) does not bind to a unique position of the SJ25C1 antibody. In some embodiments, the immunogen comprises the amino acid sequences set forth in SEQ ID NO. 28 and SEQ ID NO. 32, optionally separated by a linker (e.g., set forth in SEQ ID NO. 33). In some embodiments, the antibody identified in (b) binds to an immunogen and does not bind to an Fc domain or fragment thereof or a molecule comprising an scFv derived from an FMC63 antibody (such as an scFv comprising the amino acid sequence SEQ ID NO: 34). In some embodiments, the antibody identified in (b) binds to a target molecule comprising amino acid sequence SEQ ID NO. 28 and does not bind to a non-target molecule comprising amino acid sequence SEQ ID NO. 32 or a non-target molecule comprising amino acid sequence SEQ ID NO. 34.
In some embodiments, identifying antibodies includes identifying individual antibody clones produced by a subject using hybridoma technology, and screening the antibody clones for binding to target molecules and non-target molecules. In some embodiments, identifying the antibody comprises: (i) Isolating B cells from the spleen of the subject and fusing them with immortalized B cells to produce hybridomas; (ii) Screening hybridomas for the production of antibodies which bind to target molecules but not to non-target molecules; (iii) Antibodies from hybridomas producing antibodies that bind to the target molecule but not to non-target molecules are sequenced, thereby identifying anti-idiotype antibodies. In some embodiments, screening hybridomas includes determining binding affinity of the hybridoma antibodies to target molecules and non-target molecules.
In an exemplary embodiment, to identify an anti-idiotype antibody that recognizes a scFv portion of an exemplary anti-CD 19 Chimeric Antigen Receptor (CAR) containing an anti-CD 19scFv derived from SJ25C1, an immunogen comprising the sequences set forth below may be used:
EVKLQQSGAELVRPGSSVKISCKASGYAFSSYWMNWVKQRPGQGLEWIGQIYPGDGDTNYNGKFKGQATLTADKSSSTAYMQLSGLTSEDSAVYFCARKTISSVVDFYFDYWGQGTTVTVSSGGGGSGGGGSGGGGSDIELTQSPKFMSTSVGDRVSVTCKASQNVGTNVAWYQQKPGQSPKPLIYSATYRNSGVPDRFTGSGSGTDFTLTITNVQSKDLADYFCQQYNRYPYTSGGGTKLEIKR (SEQ ID NO: 28). In some embodiments, the immunogen may further comprise an Fc domain or fragment thereof, for example, as set forth in SEQ ID NO. 32.
In an exemplary embodiment, to identify an anti-idiotype antibody that recognizes a scFv portion of an exemplary anti-CD 19 Chimeric Antigen Receptor (CAR) containing an anti-CD 19scFv derived from FMC63, an immunogen comprising the sequences set forth below may be used:
DIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGSTSGSGKPGSGEGSTKGEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSS(SEQ ID NO:34)。
in some embodiments, the immunogen may further comprise an Fc domain or fragment thereof, for example, as set forth in SEQ ID NO. 32. In some embodiments, to identify an anti-idiotype antibody that recognizes a scFv portion of an exemplary anti-CD 19 Chimeric Antigen Receptor (CAR) containing an anti-CD 19scFv derived from FMC63, an immunogen comprising the sequences set forth below may be used:
DIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGSTSGSGKPGSGEGSTKGEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSSPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:35)。
chimeric Antigen Receptor (CAR) and genetically engineered cells
In some embodiments, the provided anti-idiotype antibodies specifically bind to an antigen binding portion of a Chimeric Antigen Receptor (CAR), such as an anti-CD 19CAR containing an antigen binding portion derived from antibody SJ25C1 or FMC 63. In some embodiments, the provided anti-idiotype antibodies bind to such CARs expressed on cells, such as cells used in conjunction with adoptive cell therapy. In some embodiments, the cell comprises one or more nucleic acids introduced by genetic engineering, thereby expressing a recombinant or genetically engineered CAR product of such nucleic acids. In some embodiments, the chimeric receptor can modulate T cell activity when genetically engineered into immune cells, and in some cases, can modulate T cell differentiation or homeostasis, thereby producing genetically engineered cells with improved in vivo longevity, survival, and/or persistence, for use, for example, in adoptive cell therapy methods. In some embodiments, the provided anti-idiotype antibodies can be used in methods of modulating one or more of these activities, including activating, stimulating, and/or expanding engineered cells expressing a target CAR.
In some embodiments, the cells comprise one or more nucleic acids introduced by genetic engineering according to the provided methods, thereby expressing recombinant or genetically engineered products of such nucleic acids. In some embodiments, the nucleic acid is heterologous, i.e., is not normally present in the cell or sample obtained from the cell, such as a nucleic acid obtained from another organism or cell, e.g., is not normally found in the cell being engineered and/or the organism from which such a cell was obtained. In some embodiments, the nucleic acid is not naturally occurring, such as nucleic acid not found in nature, including nucleic acids encoding chimeric combinations of nucleic acids from various domains of a plurality of different cell types. In a specific embodiment, the nucleic acid comprises a gene encoding a CAR.
In some embodiments, the provided methods can be performed simultaneously, sequentially or in parallel with one or more process steps for making or preparing genetically engineered cells. The processing steps of the method may include any one or more of a plurality of cell processing steps, alone or in combination. In particular embodiments, the treatment step comprises transducing or transfecting the cell with one or more nucleic acids, e.g., a heterologous polynucleotide comprising a gene encoding a recombinant receptor. In certain embodiments, the cells are transduced with viral vector particles containing, for example, a retroviral vector encoding a recombinant product for expression in the cells. In certain embodiments, the cells are transfected with one or more non-viral nucleic acids, such as episomal plasmids or transposons. The method may further and/or alternatively comprise other processing steps, such as steps for isolating, separating, selecting, washing, suspending, diluting, concentrating and/or formulating cells. In some cases, the methods may further comprise an ex vivo step of culturing, stimulating or expanding the cells (e.g., stimulating the cells, e.g., to induce their proliferation and/or activation), and in some cases, may be performed in accordance with the provided methods. In some embodiments, the methods comprise isolating cells from a subject, preparing, processing, culturing, and/or engineering them, and reintroducing them into the same subject either before or after cryopreservation.
In some embodiments, the method comprises the processing steps performed in the following order: first isolating (e.g., selecting or separating) cells, e.g., primary cells, from a biological sample; incubating the selected cells with viral vector particles for transduction; and formulating the transduced cells into a composition. In some cases, the transduced cells are activated, expanded or proliferated ex vivo, such as by stimulation in the presence of a stimulating agent, such as according to the provided methods. In some embodiments, the methods may include one or more processing steps in washing, suspending, diluting, and/or concentrating cells, which may be performed before, during, or simultaneously with or after one or more of the isolation (e.g., separation or selection), transduction, stimulation, and/or formulation steps.
In particular embodiments, the cells to be transfected or transduced are not isolated, selected or enriched prior to contact with the one or more nucleic acids. In some embodiments, the cell is not selected prior to contacting the cell with the one or more nucleic acids. In some embodiments, the cells to be transfected or transduced are not enriched prior to contacting the cells with one or more nucleic acids.
In some embodiments, the methods provided in connection with the methods, including in connection with preparing a composition containing genetically engineered cells, one or more cell processing steps in connection with preparing, processing, and/or incubating the cells may be performed in the lumen of a centrifugal chamber, such as a substantially rigid chamber, which is generally cylindrical and rotatable about a rotational axis, which provides certain advantages over other available methods. In some embodiments, all processing steps are performed in the same centrifugal chamber. In some embodiments, one or more of the processing steps are performed in different centrifugal chambers, such as multiple centrifugal chambers of the same type. Such methods include any of those described in International publication No. WO 2016/073602.
Exemplary centrifugal chambers include those produced and sold by Biosafe SA, includingAnd->2, including a-200/F and a-200 centrifugal chambers, and various kits for use with such systems. Exemplary chambers, systems, and processing instruments and cabinets are described, for example, in U.S. patent No.6,123,655, U.S. patent No.6,733,433, and published U.S. patent application publication No. US 2008/0171951, and published international patent application publication No. WO 00/38762, the respective contents of which are incorporated herein by reference in their entirety. Depending on the particular process (e.g., dilution, washing, transduction, formulation), the selection of the particular kit appropriate for the process is within the level of the skilled artisan. Exemplary kits for use with such systems include, but are not limited to, disposable reagents sold by Biosafe SA under the product names CS-430.1, CS-490.1, CS-600.1 or CS-900.2 A box.
In some embodiments, the system is included with and/or associated with other instruments, including instruments for operating, automating, controlling, and/or monitoring aspects of various process steps performed in the system. In some embodiments, the instrument is housed within a cabinet. In some embodiments, the instrument includes a cabinet including a housing containing control circuitry, a centrifuge, a lid, a motor, a pump, a sensor, a display, and a user interface. Exemplary devices are described in U.S. Pat. No.6,123,655, U.S. Pat. No.6,733,433, and U.S. 2008/0171951.
In some embodiments, the system includes a series of containers, such as bags, tubes, stopcocks, clamps, connectors, and centrifugal chambers. In some embodiments, the container (e.g., bag) comprises one or more containers (e.g., bags) containing the cells and vector particles to be transduced or transfected, e.g., viral vector particles or non-viral plasmids, in the same container or in separate containers (e.g., the same bag or separate bags). In some embodiments, the system further comprises one or more containers, such as bags, containing a medium, such as a diluent and/or wash solution, that is drawn (pull) into the chamber and/or other components during the process to dilute, re-suspend, and/or wash the components and/or compositions. The vessel may be connected at one or more locations in the system, such as at locations corresponding to the input line, diluent line, wash line, waste line, and/or output line.
In some embodiments, the system (such as a closed system) is sterile. In some embodiments, all connections of components of the system (such as between the pipeline and the vessel) via the connectors are made under sterile conditions. In some embodiments, the connection is made under laminar flow. In some embodiments, the connection is made between the tube and the container using a sterile connection device that creates a sterile connection (such as a sterile weld). In some embodiments, the sterile connection device is connected under sufficiently high thermal conditions to maintain sterility, such as a temperature of at least 200 ℃, such as at least 260 ℃ or 300 ℃.
In some embodiments, the system may be disposable, such as a disposable kit. In some embodiments, for example, in a process that occurs in a continuous or semi-continuous manner, the disposable kit can be used in multiple cycles of one or more processes, such as at least 2, 3, 4, 5, or more times. In some embodiments, the system (e.g., a disposable kit) is used to treat cells from a single patient. In aspects of the method, the process need not be performed in the same closed system, such as in the same centrifugal chamber, but may be performed under different closed systems, such as in different centrifugal chambers; in some embodiments, such different centrifugal chambers are located at corresponding points of the method associated with the same system, such as associated with the same centrifuge. In some embodiments, all of the processing steps are performed in a closed system, wherein all or a subset of each or more of the processing steps are performed in the same or different centrifugal chambers.
Target Chimeric Antigen Receptor (CAR)
In some embodiments, the provided anti-idiotype antibodies specifically bind to an extracellular domain of a target CAR that contains an antigen binding domain of an antibody or antibody fragment that provides specificity for a desired antigen (e.g., a tumor antigen) and is operably linked or linked to an intracellular signaling domain. In some embodiments, the antigen binding domain comprises antibody SJ25C1 or an antibody fragment derived from a portion of SJ25C 1. In some embodiments, the antigen binding domain comprises antibody FMC63 or an antibody fragment derived from a portion of FMC 63. In some embodiments, the intracellular signaling domain is an activated intracellular domain portion, such as a T cell activation domain, that provides a primary activation signal. In some embodiments, the intracellular signaling domain contains or otherwise contains a costimulatory signaling domain that promotes effector function.
In some embodiments, engineered cells (such as T cells) are provided that express a CAR specific for a particular antigen (or marker or ligand), such as an antigen expressed on the surface of a particular cell type. In some embodiments, the antigen is a polypeptide. In some embodiments, it is a carbohydrate or other molecule. In some embodiments, the antigen is selectively expressed or overexpressed on cells of a disease or disorder (e.g., tumor or pathogenic cells) as compared to normal or non-targeted cells or tissues. In other embodiments, the antigen is expressed on normal cells and/or on engineered cells.
In particular embodiments, the recombinant receptor (such as a chimeric receptor) contains an intracellular signaling region comprising a cytoplasmic signaling domain (also interchangeably referred to as an intracellular signaling domain), such as a cytoplasmic (intracellular) region capable of inducing a primary activation signal in a T cell, such as a cytoplasmic signaling domain of the zeta chain of a T Cell Receptor (TCR) component (e.g., a cytoplasmic signaling domain of the zeta chain of a CD3-zeta (CD 3 zeta) chain, or a functional variant or signaling portion thereof), comprising an immune receptor tyrosine activation motif (ITAM).
In some embodiments, the chimeric receptor further comprises an extracellular ligand binding domain that specifically binds to a ligand (e.g., an antigen). In some embodiments, the chimeric receptor is a CAR that contains an extracellular antigen recognition domain that specifically binds to an antigen. In some embodiments, the ligand (such as an antigen) is a protein expressed on the surface of a cell. In some embodiments, the CAR is a TCR-like CAR, and the antigen is a processed peptide antigen, such as a peptide antigen of an intracellular protein, that is recognized on the cell surface in the context of a Major Histocompatibility Complex (MHC) molecule, as with a TCR.
Exemplary antigen receptors (including CARs), and methods for engineering and introducing such receptors into cells include, for example, those described in the following documents: international patent application publication nos. WO200014257, WO2013126726, WO2012/129514, WO2014031687, WO2013/166321, WO2013/071154, WO2013/123061, U.S. patent application publication nos. US2002131960, US2013287748, US20130149337, U.S. patent nos. 6,451,995, 7,446,190, 8,252,592, 8,339,645, 8,398,282, 7,446,179, 6,410,319, 7,070,995, 7,265,209, 7,354,762, 7,446,191, 8,324,353 and 8,479,118, and european patent application nos. EP2537416, and/or Sadelain et al, cancer discover.2013 april;3 (4) 388-398; davila et al (2013) PLoS ONE 8 (4): e61338; turtle et al, curr.Opin.Immunol.,2012October;24 633-39; wu et al, cancer,2012March 18 (2): 160-75. In some aspects, antigen receptors include CARs as described in U.S. Pat. No.7,446,190, and those described in international patent application publication No. WO/2014055668 A1. Examples of CARs include those disclosed in any of the above publications, such as WO2014031687, U.S. Pat. No.8,339,645, U.S. Pat. No.7,446,179, U.S. 2013/0149337, U.S. Pat. No.7,446,190, U.S. Pat. No.8,389,282, kochenderfer et al, 2013,Nature Reviews Clinical Oncology,10,267-276 (2013); wang et al (2012) J.Immunother35 (9): 689-701; and Brentjens et al, sci Transl Med.2013 (177). See also WO2014031687, U.S. Pat. No.8,339,645, U.S. Pat. No.7,446,179, U.S. 2013/0149337, U.S. Pat. No.7,446,190 and U.S. Pat. No.8,389,282.
In some embodiments, the CAR is constructed to be specific for a particular antigen (or marker or ligand), such as an antigen expressed in a particular cell type targeted by adoptive therapy, e.g., a cancer marker and/or antigen intended to induce an inhibitory (dampling) response, such as an antigen expressed on a normal or non-diseased cell type. Thus, a CAR typically comprises one or more antigen binding molecules, such as one or more antigen binding fragments, domains, or portions, or one or more antibody variable domains and/or antibody molecules, in its extracellular portion. In some embodiments, the CAR comprises one or more antigen-binding portions of an antibody molecule, such as a single chain antibody fragment (scFv) derived from a variable heavy chain (VH) and a variable light chain (VL) of a monoclonal antibody (mAb).
In some embodiments, the antibody or antigen binding portion thereof is expressed on a cell as part of a CAR. In general, CARs containing antibodies or antigen binding fragments that exhibit TCR-like specificity for peptide-MHC complexes may also be referred to as TCR-like CARs. In some embodiments, the extracellular antigen binding domain specific for the MHC-peptide complex of the TCR-like CAR is linked in some aspects via a linker and/or transmembrane domain to one or more intracellular signaling components. In some embodiments, such molecules may mimic or approximate a signal, typically through a native antigen receptor (such as a TCR), and optionally through the binding of such a receptor to a co-stimulatory receptor.
In some embodiments, a recombinant receptor, such as a chimeric receptor (e.g., CAR), includes a ligand binding domain that binds (e.g., specifically binds) to an antigen (or ligand). Wherein the antigen targeted by the chimeric receptor is an antigen expressed in the context of a disease, disorder or cell type targeted via adoptive cell therapy. Wherein the diseases and disorders are proliferative, neoplastic and malignant diseases and disorders, including cancers and tumors, including cancers of the blood, immune system, such as lymphomas, leukemias and/or myelomas, such as B, T and myelogenous leukemias, lymphomas and multiple myelomas.
In some embodiments, the antigen (or ligand) is a polypeptide. In some embodiments, it is a carbohydrate or other molecule. In some embodiments, the antigen (or ligand) is selectively expressed or over-expressed on cells of a disease or disorder (e.g., tumor or pathogenic cells) as compared to normal or non-targeted cells or tissues. In other embodiments, the antigen is expressed on normal cells and/or on engineered cells.
In some embodiments, the CAR contains an antibody or antigen binding fragment (e.g., scFv) that specifically recognizes an antigen, such as an intact antigen expressed on the surface of a cell.
In some embodiments, the antigen (or ligand) is a tumor antigen or a cancer marker. In some embodiments, the antigen (or ligand) is αvβ6 integrin (avb 6 integrin), B Cell Maturation Antigen (BCMA), B7-H6, carbonic anhydrase 9 (CA 9, also known as CAIX or G250), cancer-testis antigen, cancer/testis antigen 1B (CTAG, also known as NY-ESO-1 and rage-2), carcinoembryonic antigen (CEA), cyclin A2, C-C motif chemokine ligand 1 (CCL-1), CD19, CD20, CD22, CD23, CD24, CD30, CD33, CD38, CD44v6, CD44v7/8, CD123, CD138, CD171, epidermal growth factor protein (EGFR), truncated epidermal growth factor protein (tgfr), type III epidermal growth factor receptor mutation (EGFR vIII), epithelial glycoprotein 2 (EPG-2), epithelial glycoprotein 40 (EPG-40), hepcidin B2, estrogen receptor A2 (ep 2), fc 5 receptor 5 (Fc-like receptor 5); also known as Fc receptor homolog 5 or FCRH 5), fetal acetylcholine receptor (fetal AchR), folic acid binding protein (FBP), folic acid receptor alpha, fetal acetylcholine receptor, ganglioside GD2, O-acetylated GD2 (OGD 2), ganglioside GD3, glycoprotein 100 (gp 100), her2/neu (receptor tyrosine kinase erbB 2), her3 (erb-B3), her4 (erb-B4), erbB dimer, human high molecular weight melanomA-Associated antigen (HMW-MAA), and, hepatitis B surface antigen, human leukocyte antigen A1 (HLA-AI), human leukocyte antigen A2 (HLa-A2), IL-22 receptor alpha (IL-22 Ra), IL-13 receptor alpha 2 (IL-13 Ra 2), kinase insertion domain receptor (kdr), kappa light chain, L1 cell adhesion molecule (L1 CAM), CE7 epitope of L1-CAM, leucine rich repeat 8 family member A (LRRC 8A), lewis Y, melanoma associated antigen (MAGE) -A1, MAGE-A3, MAGE-A6, mesothelin, c-Met, murine Cytomegalovirus (CMV), mucin 1 (MUC 1), MUC16, natural killer group 2 member D (NKG 2D) ligand, melanoma A (MART-1), neural Cell Adhesion Molecule (NCAM), carcinoembryonic antigen, melanoma preferential expression antigen (PRAME), progesterone receptor, prostate specific antigen, prostate stem Cell Antigen (CA), prostate specific membrane (prostate specific antigen), human vascular endothelial cell antigen (VEGFR) 2, human vascular receptor-specific tumor cell antigen (VEGFR 2), human vascular receptor-associated tumor antigen (VEGFR 2), vascular receptor-specific tumor antigen (R2) or tumor antigen-specific tumor growth factor (VEGFR 2), vascular antigen (tumor antigen-specific tumor antigen-2), vascular receptor (tumor antigen-specific tumor antigen-2) or tumor growth factor (tumor antigen-specific receptor-2), vascular antigen-associated protein (VEGFR 2) receptor-2 (tumor 2), tumor antigen-specific tumor antigen (tumor 2), tumor antigen-2), tumor antigen (tumor 2) receptor-tumor antigen), tumor antigen (human tumor antigen), tumor antigen-tumor antigen (human tumor antigen), vascular antigen, HPV or other pathogens and/or oncogenic forms thereof. In some embodiments, the antigen targeted by the receptor includes antigens associated with B cell malignancy, such as any of a number of known B cell markers. In some embodiments, the antigen targeted by the receptor is CD20, CD19, CD22, ROR1, CD45, CD21, CD5, CD33, igκ, igλ, CD79a, CD79b, or CD30.
In some embodiments, the antigen is a pathogen-specific antigen. In some embodiments, the antigen is a viral antigen (e.g., a viral antigen from HIV, HCV, HBV, etc.), a bacterial antigen, and/or a parasitic antigen.
In some embodiments, the antigen targeted by the receptor includes antigens associated with B cell malignancy, such as any of a number of known B cell markers. In some embodiments, the antigen targeted by the receptor is CD20, CD19, CD22, ROR1, CD45, CD21, CD5, CD33, igκ, igλ, CD79a, CD79b, or CD30. In some embodiments, the antigen is CD19 and is specifically bound by an anti-CD 19 antibody, such as SJ25C1 or an antigen binding fragment derived from SJ25C1, or FMC63 or an antigen binding fragment derived from FMC 63.
In some embodiments, the antigen binding proteins, antibodies, and antigen binding fragments thereof specifically recognize the antigen of the full length antibody. In some embodiments, the heavy and light chains of an antibody may be full length or may be antigen binding portions (Fab, F (ab') 2, fv, or single chain Fv fragments (scFv)). In other embodiments, the antibody heavy chain constant region is selected from, for example, igG1, igG2, igG3, igG4, igM, igA1, igA2, igD, and IgE, particularly selected from, for example, igG1, igG2, igG3, and IgG4, more particularly IgG1 (e.g., human IgG 1). In another embodiment, the antibody light chain constant region is selected from, for example, kappa or lambda, especially kappa.
Wherein the antibody provided is an antibody fragment. An "antibody fragment" refers to a molecule other than an intact antibody that comprises a portion of the intact antibody that binds to an antigen to which the intact antibody binds. Examples of antibody fragments include, but are not limited to Fv, fab, fab ', fab ' -SH, F (ab ') 2; a diabody; a linear antibody; variable heavy chain (VH) regions, single chain antibody molecules such as scFv and single domain VH monoclonal antibodies; and multispecific antibodies formed from antibody fragments. In particular embodiments, the antibody is a single chain antibody fragment, such as an scFv, comprising a variable heavy chain region and/or a variable light chain region.
The term "variable region" or "variable domain" refers to a domain of an antibody heavy or light chain that relates to binding of an antibody to an antigen. The variable domains of the heavy and light chains of natural antibodies (VH and VL, respectively) generally have similar structures, each domain comprising four conserved Framework Regions (FR) and three CDRs. (see, e.g., kindt et al, kuby Immunology,6th ed., W.H. Freeman and Co., page 91 (2007); individual VH or VL domains may be sufficient to confer antigen binding specificity. Furthermore, VH or VL domains from antigen-binding antibodies may be used to isolate antibodies that bind a particular antigen to screen libraries of complementary VL or VH domains, respectively. See, e.g., portolano et al, J.Immunol.150:880-887 (1993); clarkson et al, nature 352:624-628 (1991).
A single domain antibody is an antibody fragment comprising all or part of the heavy chain variable domain or all or part of the light chain variable domain of an antibody. In certain embodiments, the single domain antibody is a human single domain antibody. In some embodiments, the CAR comprises an antibody heavy chain domain that specifically binds an antigen, such as a cancer marker or a cell to be targeted (such as a tumor cell or cancer cell) or a cell surface antigen of a disease, such as any target antigen described herein or known in the art.
Antibody fragments may be produced by a variety of techniques, including, but not limited to, proteolytic digestion of intact antibodies and production by recombinant host cells. In some embodiments, the antibody is a recombinantly produced fragment, such as a fragment comprising a non-naturally occurring arrangement, such as a fragment having two or more antibody regions or chains joined by a synthetic linker (e.g., a peptide linker), and/or a fragment that cannot be produced by enzymatic digestion of a naturally occurring intact antibody. In some embodiments, the antibody fragment is an scFv.
A "humanized" antibody is one in which all or substantially all CDR amino acid residues are derived from non-human CDRs and all or substantially all FR amino acid residues are derived from human FRs. The humanized antibody optionally may comprise at least a portion of an antibody constant region derived from a human antibody. "humanized form" of a non-human antibody refers to a variant of a non-human antibody that has undergone humanization, typically in order to reduce immunogenicity to humans, while retaining the specificity and affinity of the parent non-human antibody. In some embodiments, some FR residues in a humanized antibody are substituted with corresponding residues from a non-human antibody (e.g., an antibody from which CDR residues are derived), e.g., for restoring or increasing antibody specificity or affinity.
Chimeric receptors, such as CARs, typically comprise an extracellular antigen binding domain, such as a portion of an antibody molecule (e.g., SJ25C1 or FMC 63), typically a Variable Heavy (VH) chain region and/or a Variable Light (VL) chain region of an antibody, e.g., a scFv antibody fragment.
In some embodiments, the chimeric antigen receptor comprises an extracellular portion comprising an antibody or antibody fragment. In some aspects, the chimeric antigen receptor comprises an extracellular portion comprising an antibody or fragment and an intracellular signaling domain. In some embodiments, the antibody or fragment comprises an scFv. In some embodiments, the scFv is derived from SJ25C1 and comprises the amino acid sequence set forth in SEQ ID NO. 28. In some embodiments, the scFv is derived from FMC63 and comprises the amino acid sequence set forth in SEQ ID NO. 34.
In some embodiments, a recombinant receptor, such as a CAR, e.g., an antibody module thereof, further comprises a spacer, which may be or comprise at least a portion of an immunoglobulin constant region, or a variant or modified form thereof, such as a hinge region, e.g., an IgG4 hinge region and/or a CH1/CL and/or Fc region. In some embodiments, the constant region or portion is a constant region or portion of a human IgG (such as IgG4 or IgG 1). In some aspects, portions of the constant region serve as a spacer between the antigen-recognizing component (e.g., scFv) and the transmembrane domain. The length of the spacer may provide increased cellular reactivity after antigen binding compared to the absence of the spacer. In some examples, the spacer is at or about 12 amino acids in length or no more than 12 amino acids in length. Exemplary spacers include those having at least about 10 to 229 amino acids, about 10 to 200 amino acids, about 10 to 175 amino acids, about 10 to 150 amino acids, about 10 to 125 amino acids, about 10 to 100 amino acids, about 10 to 75 amino acids, about 10 to 50 amino acids, about 10 to 40 amino acids, about 10 to 30 amino acids, about 10 to 20 amino acids, or about 10 to 15 amino acids (and including any integer between the endpoints of any of the listed ranges). In some embodiments, the spacer has about 12 amino acids or less, about 119 amino acids or less, or about 229 amino acids or less. Exemplary spacers include an IgG4 hinge alone, an IgG4 hinge linked to CH2 and CH3 domains, or an IgG4 hinge linked to a CH3 domain. In some embodiments, the spacer has the sequence set forth in SEQ ID NO. 150 and is encoded by the sequence set forth in SEQ ID NO. 151. In some embodiments, the spacer has the sequence set forth in SEQ ID NO. 152. In some embodiments, the spacer has the sequence set forth in SEQ ID NO. 153. Exemplary spacers include, but are not limited to, those described in Hudecek et al (2013) clin.cancer res.,19:3153, international patent application publication No. WO2014031687, U.S. patent No.8,822,647, or published application No. US 2014/0271635.
In some embodiments, the constant region or portion is a constant region or portion of IgD. In some embodiments, the spacer has the sequence set forth in SEQ ID NO. 154. In some embodiments, the spacer has an amino acid sequence that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to any one of SEQ ID NOs 1, 3, 4 and 5.
In some embodiments, the antigen receptor comprises an intracellular domain linked directly or indirectly to an extracellular domain. In some embodiments, the chimeric antigen receptor comprises a transmembrane domain that connects an extracellular domain and an intracellular signaling domain. In some embodiments, the transmembrane domain is fused to an extracellular domain. In some embodiments, the intracellular signaling domain comprises ITAM. For example, in some aspects, the antigen recognition domain (e.g., extracellular domain) is typically linked to one or more intracellular signaling components that mimic the signaling component of activation by an antigen receptor complex (such as a TCR complex) and/or signal via another cell surface receptor, e.g., in the case of a CAR. In some embodiments, the chimeric receptor comprises a transmembrane domain linked or fused between an extracellular domain (e.g., scFv) and an intracellular signaling domain. Thus, in some embodiments, an antigen binding component (e.g., an antibody) is linked to one or more transmembrane and intracellular signaling domains.
In one embodiment, a transmembrane domain is used that is naturally associated with one of the domains in the receptor (e.g., CAR). In some cases, the transmembrane domain is selected or modified by amino acid substitution to avoid binding of such domain to the transmembrane domain of the same or a different surface membrane protein, thereby minimizing interactions with other members of the receptor complex.
In some embodiments, the transmembrane domain is derived from a natural or synthetic source. In the case where the source is natural, in some aspects the domain is derived from any membrane-bound protein or transmembrane protein. The transmembrane regions include those derived from the alpha, beta or zeta chain of the T cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154 (i.e., at least the transmembrane region thereof). Alternatively, the transmembrane domain in some embodiments is synthetic. In some aspects, the synthetic transmembrane domain comprises predominantly hydrophobic residues, such as leucine and valine. In some aspects, triplets of phenylalanine, tryptophan, and valine will be found at each end of the synthetic transmembrane domain. In some embodiments, the attachment is by a linker, spacer, and/or transmembrane domain. In some aspects, the transmembrane domain comprises a transmembrane portion of CD 28.
In some embodiments, the extracellular domain and the transmembrane domain may be directly or indirectly linked. In some embodiments, the extracellular domain and the transmembrane are connected by a spacer, such as any of the spacers described herein. In some embodiments, the receptor contains an extracellular portion of a molecule from which the transmembrane domain is derived, such as a CD28 extracellular portion.
Wherein the intracellular signaling domains are those that mimic or approximate a signal through a natural antigen receptor, mimic or approximate a signal through the binding of such receptor to a co-stimulatory receptor, and/or mimic or approximate a signal through only a co-stimulatory receptor. In some embodiments, there is a short oligopeptide or polypeptide linker, e.g., a linker of 2 to 10 amino acids in length, such as a linker containing glycine and serine (e.g., glycine-serine duplex), and the linker forms a junction between the transmembrane domain and the cytoplasmic signaling domain of the CAR.
T cell activation is described in some aspects as mediated by two classes of cytoplasmic signaling sequences: those that initiate antigen-dependent primary activation by TCRs (primary cytoplasmic signaling sequences), and those that function in an antigen-independent manner to provide secondary signaling or costimulatory signaling (secondary cytoplasmic signaling sequences). In some aspects, the CAR comprises one or both of such signal transduction components.
Receptors (e.g., CARs) typically include at least one intracellular signaling component. In some aspects, the CAR comprises a primary cytoplasmic signaling sequence that modulates primary activation of the TCR complex. The primary cytoplasmic signaling sequence that acts in a stimulatory manner may contain a signaling motif, known as an immunoreceptor tyrosine activation motif or ITAM. Examples of ITAMs containing primary cytoplasmic signal sequences include those derived from the cd3ζ chain, fcrγ, cd3γ, cd3δ and cd3ε. In some embodiments, the cytoplasmic signaling molecule in the CAR contains a cytoplasmic signaling domain, a portion thereof, or a sequence derived from cd3ζ.
In some embodiments, the receptor comprises an intracellular component of the TCR complex, such as a TCR CD3 chain, e.g., a cd3ζ chain, that mediates T cell activation and cytotoxicity. Thus, in some aspects, the antigen binding moiety is linked to one or more cell signaling modules. In some embodiments, the cell signaling module comprises a CD3 transmembrane domain, a CD3 intracellular signaling domain, and/or other CD transmembrane domain. In some embodiments, the receptor (e.g., CAR) further comprises a portion of one or more additional molecules, such as Fc receptor gamma, CD8, CD4, CD25, or CD16. For example, in some aspects, the CAR or other chimeric receptor comprises a chimeric molecule between CD3-zeta (CD 3- ζ) or Fc receptor γ and CD8, CD4, CD25, or CD16.
In some embodiments, upon connection of the CAR or other chimeric receptor, the cytoplasmic domain or intracellular signaling domain of the receptor activates at least one of the normal effector functions or responses of an immune cell, such as a T cell engineered to express the CAR. For example, in some cases, the CAR induces a function of the T cell, such as cytolytic activity or T helper activity, such as secretion of cytokines or other factors. In some embodiments, a truncated portion of the intracellular signaling domain of an antigen receptor component or co-stimulatory molecule is used instead of the intact immunostimulatory chain, e.g., provided that it transduces an effector function signal. In some embodiments, one or more intracellular signaling domains comprise cytoplasmic sequences of T Cell Receptors (TCRs), and in some aspects also include those sequences of co-receptors that naturally cooperate with such receptors to initiate signal transduction upon antigen receptor binding, and/or any derivatives or variants of such molecules, and/or any synthetic sequences having the same functional ability.
In the case of native TCRs, complete activation typically requires not only signaling by the TCR, but also a co-stimulatory signal. Thus, in some embodiments, to facilitate complete activation, components for generating a secondary signal or co-stimulatory signal are also included in the CAR. In other embodiments, the CAR does not include a component for generating a co-stimulatory signal. In some aspects, the additional CAR is expressed in the same cell and provides a component for generating a secondary signal or a co-stimulatory signal.
T cell activation is described in some aspects as mediated by two classes of cytoplasmic signaling sequences: those that initiate antigen-dependent primary activation by TCRs (primary cytoplasmic signaling sequences), and those that function in an antigen-independent manner to provide secondary signaling or costimulatory signaling (secondary cytoplasmic signaling sequences). In some aspects, the CAR comprises one or both of such signal transduction components.
In some embodiments, the chimeric antigen receptor comprises an intracellular domain of a T cell costimulatory molecule. In some embodiments, the CAR comprises a signaling domain and/or transmembrane portion of a co-stimulatory receptor, such as CD28, 4-1BB, OX40, DAP10, and ICOS. In some aspects, the same CAR includes both an activating component and a co-stimulatory component. In some embodiments, the chimeric antigen receptor contains an intracellular domain derived from a T cell costimulatory molecule, or a functional variant thereof, such as between a transmembrane domain and an intracellular signaling domain. In some aspects, the T cell costimulatory molecule is CD28 or 41BB.
In some embodiments, the activation domain is contained within one CAR, while the co-stimulatory component is provided by another CAR that recognizes another antigen. In some embodiments, the CAR comprises an activating or stimulating CAR, a co-stimulating CAR, both expressed on the same cell (see WO 2014/055668). In some aspects, the cell comprises one or more stimulatory or activating CARs and/or co-stimulatory CARs. In some embodiments, the cells further include an inhibitory CAR (iCAR, see Fedorov et al, sci.tranl.medicine, 5 (215) (month 12 2013), such as a CAR that recognizes an antigen other than an antigen associated with and/or specific for a disease or disorder, thereby reducing or inhibiting, by binding of the inhibitory CAR to its ligand, an activation signal delivered by the disease-targeted CAR, e.g., to reduce off-target effects.
In certain embodiments, the intracellular signaling domain comprises a CD28 transmembrane and signaling domain linked to a CD3 (e.g., CD3- ζ) intracellular domain. In some embodiments, the intracellular signaling domain comprises a chimeric CD28 and CD137 (4-1 BB, TNFRSF9) co-stimulatory domain linked to a CD3 zeta intracellular domain.
In some embodiments, the CAR comprises one or more, e.g., two or more, co-stimulatory domains and an activation domain, e.g., a primary activation domain, in the cytoplasmic portion. Exemplary CARs include intracellular components of CD 3-zeta, CD28, and 4-1 BB.
In some embodiments, the antigen receptor further comprises a marker, and/or the cell expressing the CAR or other antigen receptor further comprises a surrogate marker, such as a cell surface marker, which can be used to confirm transduction or engineering of the cell expressing the receptor. In some aspects, the marker comprises all or part of a CD34, NGFR, or epidermal growth factor receptor (e.g., a truncated form), such as a truncated form of such a cell surface receptor (e.g., tgfr). In some embodiments, the nucleic acid encoding the marker is operably linked to a polynucleotide encoding a linker sequence, such as a cleavable linker sequence, e.g., T2A. For example, the marker and optional linker sequence may be any of those disclosed in published patent application number WO 2014031687. For example, the marker may be truncated EGFR (tgfr) optionally linked to a linker sequence, such as a T2A cleavable linker sequence. In embodiments, tEGFR contains the amino acid sequence set forth in SEQ ID NO 155 or 156. In some embodiments, tEGFR contains an amino acid sequence having or having about 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater than 99% sequence identity to the sequence set forth in SEQ ID NO 155 or 156.
In some embodiments, the marker is a molecule (e.g., a cell surface protein) or portion thereof that is not naturally present on a T cell or is not naturally present on the surface of a T cell. In some embodiments, the molecule is a non-self molecule, such as a non-self protein, i.e., a protein that is not recognized as "self" by the immune system of the host in which the cell is to be adoptively transferred.
In some embodiments, the marker has no therapeutic function and/or no effect other than being used as a marker of genetic engineering (e.g., for selecting successfully engineered cells). In other embodiments, the marker may be a therapeutic molecule or a molecule that otherwise performs some desired function, such as a ligand of a cell that will be encountered in vivo, such as a co-stimulatory or immune checkpoint molecule that enhances and/or inhibits a cellular response upon adoptive transfer and encountering the ligand.
In some cases, the CAR is referred to as a first, second, and/or third generation CAR. In some aspects, the first generation CAR is a CAR that provides only CD3 chain-induced signaling after antigen binding; in some aspects, the second generation CAR is a CAR that provides such signals and co-stimulatory signals, such as a CAR comprising an intracellular signaling domain from a co-stimulatory receptor such as CD28 or CD 137; in some aspects, the third generation CAR is a CAR comprising multiple co-stimulatory domains of different co-stimulatory receptors.
In some embodiments, the chimeric antigen receptor comprises an extracellular portion comprising an antibody or fragment described herein. In some aspects, the chimeric antigen receptor comprises an extracellular portion comprising an antibody or fragment described herein and an intracellular signaling domain. In some embodiments, the antibody or fragment comprises scFv or single domain V H Antibodies, and intracellular domains contain ITAM. In some aspects, the intracellular signaling domain comprises a signaling domain of the zeta chain of the CD3-zeta (CD 3 zeta) chain. In some embodiments, the chimeric antigen receptor comprises a transmembrane domain disposed between an extracellular domain and an intracellular signal transduction region.
In some aspects, the transmembrane domain comprises a transmembrane portion of CD 28. The extracellular domain and the transmembrane may be directly or indirectly linked. In some embodiments, the extracellular domain and the transmembrane are connected by a spacer, such as any of the spacers described herein. In some embodiments, the chimeric antigen receptor comprises an intracellular domain of a T cell costimulatory molecule, such as between a transmembrane domain and an intracellular signaling domain. In some aspects, the T cell costimulatory molecule is CD28 or 4-1BB.
For example, in some embodiments, the CAR comprises an antibody (e.g., an antibody fragment), a transmembrane domain that is or comprises a transmembrane portion of CD28 or a functional variant thereof, and an intracellular signaling domain comprising a signaling portion of CD28 or a functional variant thereof and a signaling portion of cd3ζ or a functional variant thereof. In some embodiments, the CAR contains an antibody (e.g., an antibody fragment), a transmembrane domain that is or contains a transmembrane portion of CD28 or a functional variant thereof, and an intracellular signaling domain that contains a signaling portion of 4-1BB or a functional variant thereof and a signaling portion of cd3ζ or a functional variant thereof. In some such embodiments, the receptor further comprises a spacer comprising a portion of an Ig molecule (such as a human Ig molecule) (such as an Ig hinge, e.g., an IgG4 hinge), such as a hinge-only spacer.
In some embodiments, the transmembrane domain of a receptor (e.g., CAR) is the transmembrane domain of human CD28 or a variant thereof, e.g., the 27-amino acid transmembrane domain of human CD28 (accession number: P10747.1), or is a transmembrane domain comprising the amino acid sequence set forth in SEQ ID No. 157 or an amino acid sequence exhibiting at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID No. 157; in some embodiments, the portion of the recombinant receptor comprising the transmembrane domain comprises the amino acid sequence set forth in SEQ ID NO 158 or an amino acid sequence having at least or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity thereto.
In some embodiments, the chimeric antigen receptor comprises an intracellular domain of a T cell costimulatory molecule. In some aspects, the T cell costimulatory molecule is CD28 or 4-1BB.
In some embodiments, the intracellular signaling region comprises an intracellular co-stimulatory signaling domain of human CD28 or a functional variant or portion thereof, such as a 41 amino acid domain thereof and/or a domain having a LL to GG substitution, such as at positions 186-187 of the native CD28 protein. In some embodiments, the intracellular signaling domain may comprise the amino acid sequence set forth in SEQ ID NO 159 or 160 or an amino acid sequence exhibiting at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO 159 or 160. In some embodiments, the intracellular region comprises the intracellular co-stimulatory signaling domain of 4-1BB or a functional variant or portion thereof, such as the 42-amino acid cytoplasmic domain of human 4-1BB (accession No. Q07011.1) or a functional variant or portion thereof, such as the amino acid sequence set forth in SEQ ID NO:161 or an amino acid sequence exhibiting at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 161.
In some embodiments, the intracellular signaling region comprises the human CD3 chain optionally a human cd3ζ -stimulating signaling domain or functional variant thereof, such as the 112AA cytoplasmic domain or the CD3 ζ signaling domain of isoform 3 of human cd3ζ (accession number: P20963.2), as described in U.S. Pat. No.7,446,190 or U.S. Pat. No.8,911,993. In some embodiments, the intracellular signal transduction region comprises the amino acid sequence set forth in SEQ ID No. 162, 163, or 164 or an amino acid sequence exhibiting at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID No. 162, 163, or 164.
In some aspects, the spacer contains only hinge regions of IgG, such as only hinges of IgG4 or IgG1, such as the hinge-only spacers set forth in SEQ ID NO. 150. In other embodiments, the spacer is an Ig hinge, e.g., with C H 2 and/or C H 3 domain linked IgG4 hinge. In some embodiments, the spacer is an Ig hinge, e.g., with C H 2 and C H 3 domain linked IgG4 hinge, such as set forth in SEQ ID NO. 152. In some embodiments, the spacer is an Ig hinge, e.g., with C alone H 3 domain linked IgG4 hinge, such as set forth in SEQ ID NO. 153. In some embodiments, the spacer is or comprises a glycine-serine rich sequence or other flexible linker, such as known flexible linkers.
For example, in some embodiments, the CAR comprises an antibody (such as an antibody fragment), including an scFv; spacers, such as spacers comprising a portion of an immunoglobulin molecule (such as a hinge region and/or one or more constant regions of a heavy chain molecule), such as spacers comprising an Ig-hinge; a transmembrane domain comprising all or part of a CD 28-derived transmembrane domain; a CD 28-derived intracellular signaling domain; and a CD3 zeta signaling domain. In some embodiments, the CAR comprises an antibody or fragment (such as an scFv), a spacer (such as any spacer comprising an Ig hinge), a CD 28-derived transmembrane domain, a 4-1 BB-derived intracellular signaling domain, and a CD3 zeta-derived signaling domain.
In some embodiments, the nucleic acid molecule encoding such a CAR construct further comprises a sequence encoding a T2A ribosome-hopping element and/or a tgfr sequence, for example downstream of the sequence encoding the CAR. In some embodiments, T cells expressing an antigen receptor (e.g., CAR) can also be generated to express truncated EGFR (EGFRt) as a non-immunogenic selection epitope (e.g., by introducing a construct encoding CAR and EGFRt separated by a T2A ribosomal switch, both proteins being expressed from the same construct), which can then be used as a marker for detecting such cells (see, e.g., U.S. patent No.8,802,374). In some embodiments, a single promoter may direct expression of RNAs that contain two or three genes in a single Open Reading Frame (ORF) (e.g., encoding molecules involved in regulating metabolic pathways and encoding recombinant receptors) separated from each other by sequences encoding self-cleaving peptides (e.g., 2A sequences) or protease recognition sites (e.g., furin). Thus, the ORF encodes a single polypeptide that is processed into separate proteins during translation (in the case of 2A) or later. In some cases, peptides such as T2A may cause the synthesis of a ribosome skip (ribosome skip) peptide bond at the C-terminus of the 2A element, resulting in separation between the end of the 2A sequence and the next downstream peptide (see, e.g., de Felipe. Genetic Vaccines and Ther.2:13 (2004) and de Felipe et al Traffic 5:616-626 (2004)). Many 2A elements are known in the art. Examples of 2A sequences that can be used in the methods and nucleic acids disclosed herein include, but are not limited to, 2A sequences from the following viruses: foot-and-mouth disease virus (F2A, e.g., SEQ ID NO: 131), equine rhinitis virus (E2A, e.g., SEQ ID NO: 130), leptopetalum album beta tetrazoma virus (T2A, e.g., SEQ ID NO:126 or 127), and porcine teschovirus-1 (P2A, e.g., SEQ ID NO:128 or 129), as described in U.S. patent publication No. 20070116690.
Recombinant receptors (such as CARs) expressed by cells administered to a subject typically recognize or specifically bind molecules that are expressed in, associated with, and/or specific for the disease or disorder being treated or cells thereof. Upon specific binding to a molecule, e.g., an antigen, the receptor typically delivers an immunostimulatory signal (such as an ITAM-transduced signal) into the cell, thereby facilitating an immune response targeted to the disease or disorder. For example, in some embodiments, the cell expresses a CAR that specifically binds to an antigen expressed by or associated with a cell or tissue of a disease or disorder. The receptor may be another receptor, such as an immunosuppressive receptor or a co-stimulatory signaling receptor, such as a CCR or iCAR or a non-signaling receptor, for example, which is used to deplete or eliminate cells using antibodies.
Genetically engineered cells and methods of producing cells
Wherein the cell expressing the chimeric antigen receptor is an engineered cell. Genetic engineering generally involves introducing nucleic acids encoding recombinant or engineered components into a composition containing cells, such as by retroviral transduction, transfection or transformation. Various methods for introducing genetically engineered components (e.g., recombinant receptors, such as CARs) are well known and may be used. Exemplary methods include methods for transferring nucleic acids encoding a receptor, including by means of viruses (e.g., retrovirus or lentivirus), transduction, transposons, and electroporation.
Vectors and methods for genetic engineering
Also provided are one or more polynucleotides (e.g., nucleic acid molecules) encoding the Chimeric Antigen Receptor (CAR), vectors for genetically engineering cells to express such CAR, and methods for producing the engineered cells. In some embodiments, the vector contains a nucleic acid encoding a CAR. In some cases, the vector is a viral vector, such as a retroviral vector, e.g., a lentiviral vector or a gamma-retroviral vector.
In some embodiments, recombinant nucleic acid is transferred into cells using recombinant infectious viral particles, e.g., vectors derived from monkey virus 40 (SV 40), adenovirus, adeno-associated virus (AAV). In some embodiments, recombinant lentiviral vectors or retroviral vectors (such as gamma-retroviral vectors) are used to transfer recombinant nucleic acids into T cells (see, e.g., koste et al (2014) Gene Therapy 2014Apr 3.doi:10.1038/gt.2014.25; carlens et al (2000) Exp Hematol28 (10): 1137-46; alonso-Camino et al (2013) Mol Ther Nucl Acids 2, e93; park et al, trends Biotechnol.2011November 29 (11): 550-557).
In some embodiments, the retroviral vector has a Long Terminal Repeat (LTR), such as a retroviral vector derived from moloney murine leukemia virus (MoMLV), myeloproliferative sarcoma virus (MPSV), murine embryonic stem cell virus (MESV), murine Stem Cell Virus (MSCV), spleen Focus Forming Virus (SFFV), or adeno-associated virus (AAV). Most retroviral vectors are derived from murine retroviruses. In some embodiments, retroviruses include those derived from any avian or mammalian cell source. Retroviruses are often amphotropic, meaning that they are capable of infecting host cells of several species, including humans. In one embodiment, the gene to be expressed replaces retroviral gag, pol and/or env sequences. A number of illustrative retroviral systems have been described (e.g., U.S. Pat. No.5,219,740;6,207,453;5,219,740; miller and Rosman (1989) BioTechniques 7:980-990; miller, A.D. (1990) Human Gene Therapy 1:5-14; scarpa et al (1991) Virology 180:849-852; burns et al (1993) Proc.Natl. Acad. Sci. USA 90:8033-8037; and Boris-Lawrie and Temin (1993) Cur. Opin. Genet. Development. 3:102-109).
Methods of lentiviral transduction are known. Exemplary methods are described, for example, in Wang et al (2012) J.Immunother.35 (9): 689-701; cooper et al (2003) blood.101:1637-1644; verhoeyen et al (2009) Methods Mol biol.506:97-114; and Cavalieri et al (2003) blood.102 (2): 497-505.
In some embodiments, the recombinant nucleic acid is transferred into T cells by electroporation (see, e.g., chicaybam et al (2013) PLoS ONE 8 (3): e60298 and Van Tedeloo et al (2000) Gene Therapy 7 (16): 1431-1437). In some embodiments, the recombinant nucleic acid is transferred into T cells by transposition (see, e.g., manuri et al (2010) Hum Gene Ther 21 (4): 427-437; shalma et al (2013) Molec Ther Nucl Acids, e74; and Huang et al (2009) Methods Mol Biol 506: 115-126). Other methods of introducing and expressing genetic material in immune cells include calcium phosphate transfection (e.g., as described in Current Protocols in Molecular Biology, john Wiley & Sons, new york.n.y.), protoplast fusion, cationic liposome-mediated transfection; tungsten particle-promoted microprojectile bombardment (Johnston, nature,346:776-777 (1990)); and strontium phosphate DNA co-precipitation (Brash et al mol. Cell biol.,7:2031-2034 (1987)).
Other methods and vectors for transferring nucleic acids encoding recombinant products are those described, for example, in International patent application publication No. WO2014055668 and U.S. Pat. No.7,446,190.
In some embodiments, cells (e.g., T cells) may be transfected with, for example, a T Cell Receptor (TCR) or Chimeric Antigen Receptor (CAR) during or after expansion. Such transfection of the gene for introduction of the desired receptor may be performed using, for example, any suitable retroviral vector. The genetically modified cell population may then be released from the initial stimulus (e.g., anti-CD 3/anti-CD 28 stimulus) and subsequently stimulated with a second type of stimulus (e.g., via a de novo introduced receptor). Such a second type of stimulus may include antigenic stimulus in the form of a peptide/MHC molecule, a cognate (cross-linked) ligand of a genetically introduced receptor (e.g., the natural ligand of a CAR), or any ligand (such as an antibody) that binds directly in-frame with a new receptor (e.g., by recognizing a constant region within the receptor). See, e.g., cheadle et al, "Chimeric antigen receptors for T-cell based therapy" Methods Mol biol.2012;907:645-66 or Barrett et al Chimeric Antigen Receptor Therapy for Cancer Annual Review of Medicine Vol.65:333-347 (2014). In some embodiments, the cells are stimulated with the provided anti-idiotype antibodies according to the provided methods.
In some cases, vectors may be used that do not require activating cells (e.g., T cells). In some such cases, the cells may be selected and/or transduced prior to activation. Thus, the cells may be engineered before or after, and in some cases simultaneously with, or during at least a portion of, the culturing.
In further nucleic acids, for example, the introduced gene is a gene that improves the efficacy of the treatment, such as by promoting the viability and/or function of the transferred cells; genes that provide genetic markers for selecting and/or assessing cells (such as assessing survival or localization in vivo); genes that improve safety, for example, by making cells susceptible to in vivo negative selection, such as Lupton s.d. et al, mol.and Cell biol.,11:6 (1991); and Riddell et al, human Gene Therapy 3:319-338 (1992); see also PCT/US91/08442 and PCT/US94/05601 to Lupton et al which describe the use of bifunctional selection fusion genes derived by fusion of a dominant positive selection marker with a negative selection marker. See, e.g., riddell et al, U.S. patent No.6,040,177, columns 14-17.
Preparation of cells and cells for genetic engineering
Provided herein are cells, including engineered cells comprising Chimeric Antigen Receptors (CARs). Also provided are populations of such cells and compositions containing such cells. Wherein the composition is an input composition comprising cells, wherein one or more cells are known or likely to or will express a recombinant receptor that is capable of being recognized or bound by a binding molecule present on one or more particles that are incubated or contacted with the cells. And wherein the composition is a composition produced by the provided methods, including output compositions containing stimulated or expanded cells therein, including compositions enriched for cells containing recombinant receptors bound or recognized by binding molecules on particles, such as wherein cells expressing recombinant receptors (e.g., chimeric receptors) constitute at least 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more of the total cells or cells of a certain type (e.g., T cells or cd8+ or cd4+ cells) in the composition. Thus, genetically engineered cells are provided that express recombinant receptors, such as CARs.
Wherein these compositions are pharmaceutical compositions and administration formulations, such as for adoptive cell therapy. Methods for engineering, producing, or generating such cells, therapeutic methods for administering the cells and compositions to a subject (e.g., patient), and methods for detecting, selecting, isolating, or separating such cells are also provided.
In some embodiments, the nucleic acid is heterologous, i.e., is not normally present in the cell or sample obtained from the cell, such as a nucleic acid obtained from another organism or cell, e.g., is not normally found in the cell being engineered and/or the organism from which such a cell was obtained. In some embodiments, the nucleic acid is not naturally occurring, such as nucleic acid not found in nature, including nucleic acids encoding chimeric combinations of nucleic acids from various domains of a plurality of different cell types.
The cells are generally eukaryotic cells, such as mammalian cells, and are typically human cells. In some embodiments, the cells are derived from blood, bone marrow, lymph or lymphoid organs, are cells of the immune system, such as cells of the innate or adaptive immunity, e.g., myeloid cells or lymphoid cells, including lymphocytes, typically T cells and/or NK cells. Other exemplary cells include stem cells, such as pluripotent and multipotent stem cells, including induced pluripotent stem cells (ipscs).
The cells are typically primary cells, such as cells isolated directly from the subject and/or isolated from the subject and frozen. In some embodiments, the cells include one or more subpopulations of T cells or other cell types, such as whole T cell populations, cd4+ cells, cd8+ cells, and subpopulations thereof, such as those defined by function, activation state, maturity, differentiation potential, expansion, recycling, localization and/or persistence, antigen specificity, antigen receptor type, presence in a particular organ or compartment, marker or cytokine secretion profile, and/or degree of differentiation. The cells may be allogeneic and/or autologous with reference to the subject to be treated. Wherein these methods include off-the-shelf methods. In some aspects, such as for off-the-shelf technology, the cells are pluripotent and/or multipotent, such as stem cells, such as induced pluripotent stem cells (ipscs). In some embodiments, the methods comprise isolating cells from a subject, preparing, processing, culturing, and/or engineering them, and reintroducing them into the same subject either before or after cryopreservation.
Wherein the T cells and/or CD4+ and/or CD8+ T cells are of the subtype and subpopulation of naive T (T) N ) Cells, effector T cells (T EFF ) Memory T cells and subtypes thereof, such as stem cell memory T (T SCM ) Central memory T (T) CM ) Effect memory T (T) EM ) Or terminally differentiated effector memory T cells, tumor Infiltrating Lymphocytes (TILs), immature T cells, mature T cells, helper T cells, cytotoxic T cells, mucosa-associated invariant T (MAIT) cells, naturally occurring and adapted regulatory T (Treg) cells, helper T cells, such as TH1 cells, TH2 cells, TH3 cells, TH17 cells, TH9 cells, TH22 cells, follicular helper T cells, alpha/beta T cells, and delta/gamma T cells.
In some embodiments, the cell is a Natural Killer (NK) cell. In some embodiments, the cells are monocytes or granulocytes, e.g., myeloid cells, macrophages, neutrophils, dendritic cells, mast cells, eosinophils, and/or basophils.
In some embodiments, the cells comprise one or more nucleic acids introduced by genetic engineering, whereby recombinant or genetically engineered products of such nucleic acids are expressed. In some embodiments, the nucleic acid is heterologous, i.e., is not normally present in the cell or sample obtained from the cell, such as a nucleic acid obtained from another organism or cell, e.g., is not normally found in the cell being engineered and/or the organism from which such a cell was obtained. In some embodiments, the nucleic acid is not naturally occurring, such as nucleic acid not found in nature, including nucleic acids encoding chimeric combinations of nucleic acids from various domains of a plurality of different cell types.
In some embodiments, the preparation of the engineered cells includes one or more culturing and/or preparation steps. Cells for introducing nucleic acid encoding a transgenic receptor (e.g., CAR) can be isolated from a sample, such as a biological sample, e.g., a sample obtained or derived from a subject. In some embodiments, the subject from which the cells are isolated is a subject having a disease or disorder or in need of or to whom cell therapy is to be administered. In some embodiments, the subject is a human in need of a particular therapeutic intervention, such as adoptive cell therapy, wherein the cells are isolated, treated, and/or engineered.
Thus, in some embodiments, the cell is a primary cell, such as a primary human cell. Samples include tissues, fluids, and other samples taken directly from a subject, as well as samples produced by one or more processing steps such as isolation, centrifugation, genetic engineering (e.g., transduction with viral vectors), washing, and/or incubation. The biological sample may be a sample obtained directly from a biological source or a treated sample. Biological samples include, but are not limited to, body fluids such as blood, plasma, serum, cerebrospinal fluid, synovial fluid, urine and sweat, tissue and organ samples, including treated samples derived therefrom.
In some aspects, the sample from which the cells are derived or isolated is blood or a blood-derived sample, or is derived from a blood component apheresis or leukocyte apheresis product. Exemplary samples include whole blood, peripheral Blood Mononuclear Cells (PBMCs), leukocytes, bone marrow, thymus, tissue biopsies, tumors, leukemia, lymphomas, lymph nodes, intestine-associated lymphoid tissue, mucosa-associated lymphoid tissue, spleen, other lymphoid tissue, liver, lung, stomach, intestine, colon, kidney, pancreas, breast, bone, prostate, cervix, testes, ovaries, tonsils or other organs, and/or cells derived therefrom. In the context of cell therapies, such as adoptive cell therapies, samples include samples from autologous and allogeneic sources.
In some embodiments, the cells are derived from a cell line, such as a T cell line. In some embodiments, the cells are obtained from a heterologous source, e.g., from mice, rats, non-human primates, and pigs.
In some embodiments, the separation of cells includes one or more preparation steps and/or non-affinity based cell separation steps. In some examples, cells are washed, centrifuged, and/or incubated in the presence of one or more reagents, e.g., to remove unwanted components, enrich for desired components, lyse, or remove cells sensitive to a particular reagent. In some examples, the cells are isolated based on one or more properties, such as density, adhesion properties, size, sensitivity to a particular component, and/or resistance.
In some examples, cells of circulating blood from the subject are obtained, for example, by apheresis or leukoapheresis. In some aspects, the sample contains lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated leukocytes, erythrocytes, and/or platelets, and in some aspects contains cells other than erythrocytes and platelets. In some embodiments, the sample or cells in the sample may be left to stand or remain prior to further processing steps prior to selection and/or enrichment of the cells. In some embodiments, the sample is maintained or maintained at a temperature of from or about 2 ℃ to 8 ℃ for at most 48 hours, such as at most 12 hours, 24 hours, or 36 hours. In certain embodiments, the cells are not selected and/or enriched prior to contacting the cells with one or more nucleic acids. In some embodiments, the sample or cell may be left to stand or remain prior to contacting or incubating the cell with the one or more nucleic acids. In certain embodiments, the sample is maintained or maintained at a temperature of from or about 2 ℃ to 8 ℃ for a period of up to 48 hours, such as up to 12 hours, 24 hours, or 36 hours, and then the cells are contacted or incubated with one or more nucleic acids.
In some embodiments, blood cells collected from a subject are washed, e.g., to remove plasma fractions, and the cells are placed in an appropriate buffer or medium for subsequent processing steps. In some embodiments, the cells are washed with Phosphate Buffered Saline (PBS). In some embodiments, the wash solution lacks calcium and/or magnesium and/or many or all divalent cations. In some aspects, the washing step is accomplished using a semi-automatic "direct current" centrifuge (e.g., cobe 2991 cell processor, baxter) according to manufacturer's instructions. In some aspects, the washing step is accomplished by Tangential Flow Filtration (TFF) according to manufacturer's instructions. In some embodiments, cells are resuspended in various biocompatible buffers such as Ca-free after washing ++ /Mg ++ Is not shown in the above (B) in PBS. In certain embodiments, components of the blood cell sample are removed and the cells are resuspended directly in culture medium.
In some embodiments, the methods include density-based cell separation methods, such as the preparation of leukocytes from peripheral blood by lysing erythrocytes and centrifuging by Percoll or Ficoll gradient.
In some embodiments, the isolation method comprises isolating different cell types based on the expression or presence of one or more specific molecules (such as surface markers, e.g., surface proteins, intracellular markers, or nucleic acids) in the cell. In some embodiments, any known separation method based on such markers may be used. In some embodiments, the separation is affinity-based or immunoaffinity-based separation. For example, in some aspects, isolating includes isolating cells and cell populations based on expression of the cells or expression levels of one or more markers (typically cell surface markers), e.g., by incubating with an antibody or binding partner that specifically binds to such markers, followed by a washing step and separating cells that have bound to the antibody or binding partner from cells that have not bound to the antibody or binding partner.
Such isolation steps may be based on positive selection wherein cells that have bound reagents are retained for further use, and/or negative selection wherein cells that have not bound antibodies or binding partners are retained. In some examples, both portions are reserved for further use. In some aspects, negative selection may be particularly useful in the absence of antibodies that can be used to specifically identify cell types in a heterogeneous population, such that isolation is optimally performed based on markers expressed by cells other than the desired population.
Isolation does not necessarily result in 100% enrichment or removal of a particular cell population or cells expressing a particular marker. For example, positive selection or enrichment of a particular type of cell (such as a cell expressing a marker) refers to increasing the number or percentage of such cells, but does not necessarily result in the complete absence of cells that do not express the marker. Likewise, negative selection, removal or depletion of a particular type of cell (such as a cell expressing a marker) refers to a reduction in the number or percentage of such cells, but does not necessarily result in complete removal of all such cells.
In some examples, multiple rounds of separation steps are performed, wherein fractions from positive or negative selection of one step are subjected to another separation step, such as subsequent positive or negative selection. In some examples, a single isolation step can simultaneously deplete cells expressing multiple markers, such as by incubating the cells with multiple antibodies or binding partners, each of which is specific for a marker that targets negative selection. Likewise, positive selection of multiple cell types can be performed simultaneously by incubating the cells with multiple antibodies or binding partners expressed on the various cell types.
For example, in some aspects, specific T cell subsets, such as cells that are positive or express high levels of one or more surface markers, e.g., CD28, are isolated by positive or negative selection techniques + 、CD62L + 、CCR7 + 、CD27 + 、CD127 + 、CD4 + 、CD8 + 、CD45RA + And/or CD45RO + T cells.
For example, a CD may be used3/CD28 conjugated magnetic beads (e.g.,m-450CD3/CD28T Cell Expander) for CD3 + 、CD28 + Positive selection of T cells. In specific embodiments, the cells are conjugated to anti-CD 3/anti-CD 28 magnetic beads (e.g., ∈10,>m-450CD3/CD28T Cell Expander) to amplify CD3 + 、CD28 + T cells. In certain embodiments, the cells are not contacted with the anti-CD 3/anti-CD 28 conjugated magnetic beads prior to contacting the cells with the one or more nucleic acids.
In some embodiments, the isolation is performed by enriching a specific cell population by positive selection, or depleting a specific cell population by negative selection. In some embodiments, positive or negative selection is accomplished by incubating the cells with one or more antibodies or other binding agents that are expressed on the positively or negatively selected cells (markers + ) Or at higher levels (markers) High height ) Specifically binds to one or more surface markers of (a).
In some embodiments, T cells are isolated from a PBMC sample by negative selection of a marker (such as CD 14) expressed on non-T cells (such as B cells, monocytes or other leukocytes). In some aspects, CD4 is used + Or CD8 + Selection step to isolate CD4 + Helper cells and CD8 + Cytotoxic T cells. Such CD4 may be selected by positive or negative selection of markers expressed or expressed to a relatively high extent on one or more naive, memory and/or effector T cell subsets + And CD8 + The population is further classified into subgroups.
In some embodiments, the antigen is selected from CD8, e.g., by positive or negative selection based on a surface antigen associated with the corresponding subpopulation + Further enrichment or depletion of cells of naive, central memoryEffector memory and/or central memory stem cells. In some embodiments, central memory T (T CM ) Enrichment of cells to increase efficacy, such as to improve long-term survival, expansion, and/or implantation after administration, is particularly robust in some aspects in such subpopulations. See Terakura et al (2012) blood.1:72-82; wang et al (2012) J Immunother.35 (9): 689-701. In some embodiments, the combination is enriched in T CM CD8 of (C) + T cells and CD4 + T cells further enhance potency.
In embodiments, memory T cells are present in CD8 + CD62L of peripheral blood lymphocytes + And CD62L - In the subgroup. For example, CD62L can be enriched or depleted from PBMC using anti-CD 8 and anti-CD 62L antibodies - CD8 + And/or CD62L + CD8 + And (3) a fraction.
In some embodiments, central memory T (T CM ) Enrichment of cells is based on positive or high surface expression of CD45RO, CD62L, CCR, CD28, CD3 and/or CD 127; in certain aspects, it is based on negative selection of cells expressing or highly expressing CD45RA and/or granzyme B. In some aspects, the enrichment of T is performed by depletion of cells expressing CD4, CD14, CD45RA, positive selection or enrichment of cells expressing CD62L CM CD8 of cells + Isolation of the population. In one aspect, central memory T (T) is performed starting with a negative cell fraction selected based on CD4 expression CM ) Enrichment of cells the negative cell fraction was subjected to negative selection based on CD14 and CD45RA expression and positive selection based on CD 62L. In some aspects, such selection is made simultaneously, and in other aspects, sequentially in either order. In some aspects, for the preparation of CD8 + The same selection procedure based on CD4 expression of cell populations or subpopulations is also used to generate CD4 + A population or subpopulation of cells such that both the positive and negative fractions from the CD4 based separation are retained and used in subsequent steps of these methods, optionally followed by one or more additional positive or negative selection steps.
In particular examples, the PBMC sample or other leukocyte sample is subjected to CD4 + Cell selection, wherein both the negative and positive fractions are retained. The negative fractions are then negative selected based on the expression of CD14 and CD45RA or CD19, and positive selection based on the marker characteristics of central memory T cells (e.g., CD62L or CCR 7), wherein positive and negative selection are performed in either order.
CD4 is detected by identifying a population of cells having a cell surface antigen + T helper cells are classified as naive, central memory and effector cells. CD4 can be obtained by standard methods + Lymphocytes. In some embodiments, naive CD4 + T lymphocytes are CD45RO - 、CD45RA + 、CD62L + 、CD4 + T cells. In some embodiments, the central memory is CD4 + The cells were CD62L + And CD45RO + A kind of electronic device. In some embodiments, the effect is CD4 + The cells were CD62L - And CD45RO - A kind of electronic device.
In one example, to enrich for CD4 by negative selection + The mixture of monoclonal antibodies typically includes antibodies directed against CD14, CD20, CD11b, CD16, HLA-DR, and CD 8. In some embodiments, the antibody or binding partner is bound to a solid support or matrix (such as magnetic or paramagnetic beads) to allow isolation of cells for positive and/or negative selection. For example, in some embodiments, immunomagnetic (or affinity magnetic) separation techniques are used (at Methods in Molecular Medicine, vol.58: metastasis Research Protocols, vol.2: cell Behavior In Vitro and In Vivo, pages 17-25, editions: S.A.Brooks and U.S. Schumacher) Humana Press inc., totowa, NJ) to separate or isolate cells and cell populations.
In some aspects, a sample or composition of cells to be isolated is incubated with a small magnetizable or magnetically responsive material, such as magnetically responsive particles or microparticles, such as paramagnetic beads (e.g., dynalbeads or MACS beads). The magnetically responsive material (e.g., particles) is typically directly or indirectly attached to a binding partner (e.g., an antibody) that specifically binds to a molecule (e.g., a surface marker) present on a cell, cells, or cell population that is desired to be isolated (e.g., desired to be negatively or positively selected).
In some embodiments, the magnetic particles or beads comprise magnetically responsive material bound to a specific binding member (such as an antibody or other binding partner). There are many well known magnetically responsive materials used in magnetic separation processes. Suitable magnetic particles include those described in U.S. Pat. No.4,452,773 to Molday and European patent Specification EP 452342B, which are incorporated herein by reference. Colloid-sized particles (such as those described in U.S. Pat. No.4,795,698 to Owen and U.S. Pat. No.5,200,084 to Liberti et al) are other examples.
Incubation is typically performed under conditions whereby an antibody or binding partner or molecule (such as a secondary antibody or other reagent) specifically binds to a cell surface molecule (if present on a cell in the sample) and such an antibody or binding partner attached to a magnetic particle or bead.
In some aspects, placing the sample in a magnetic field, those cells having magnetically responsive or magnetizable particles attached thereto will be attracted to the magnet and separate from unlabeled cells. For positive selection, cells attracted to the magnet were retained; for negative selection, cells that were not attracted (unlabeled cells) were retained. In some aspects, a combination of positive and negative selections is performed during the same selection step, wherein the positive and negative fractions are retained and further processed or subjected to further separation steps.
In certain embodiments, the magnetically responsive particles are coated in a primary antibody or other binding partner, a secondary antibody, lectin, enzyme, or streptavidin. In certain embodiments, the magnetic particles are attached to the cells by coating with primary antibodies specific for one or more markers. In certain embodiments, cells are labeled with a primary antibody or binding partner rather than beads, and then magnetic particles coated with a cell type specific secondary antibody or coated with other binding partners (e.g., streptavidin) are added. In certain embodiments, streptavidin-coated magnetic particles are used in combination with biotinylated primary or secondary antibodies.
In some embodiments, the magnetically responsive particles remain attached to cells that are subsequently incubated, cultured, and/or engineered; in some aspects, the particles are attached to cells for administration to a patient. In some embodiments, the magnetizable or magnetically responsive particles are removed from the cells. Methods of removing magnetizable particles from cells are known, including, for example, the use of competitive non-labeled antibodies, and magnetizable particles or antibodies conjugated with cleavable linkers. In some embodiments, the magnetizable particles are biodegradable.
In some embodiments, affinity-based selection is performed by Magnetic Activated Cell Sorting (MACS) (Miltenyi Biotec, auburn, CA). Magnetically Activated Cell Sorting (MACS) systems are capable of selecting cells having magnetized particles attached thereto in high purity. In certain embodiments, MACS operates in a mode in which non-target substances and target substances are eluted sequentially after application of an external magnetic field. That is, cells attached to the magnetized particles remain in place while unattached material is eluted. Then, after this first elution step is completed, the substances that are trapped in the magnetic field and are protected from elution are released in such a way that they can be eluted and recovered. In certain embodiments, non-target cells are labeled and removed from the heterogeneous cell population.
In certain embodiments, the separation or isolation is performed using a system, apparatus or device that performs one or more of the separation, cell preparation, isolation, treatment, incubation, culture, and/or formulation method steps. In some aspects, the system is used to perform each of these steps in a closed or sterile environment, for example, to minimize errors, user handling, and/or contamination. In one example, the system is a system as described in international patent application publication No. WO2009/072003 or US20110003380 A1. In one example, the system is a system as described in International publication No. WO 2016/073602.
In some embodiments, the system or apparatus performs one or more (e.g., all) of the separating, processing, engineering, and formulation steps in an integrated or self-contained system and/or in an automated or programmable manner. In some aspects, the system or apparatus includes a computer and/or computer program in communication with the system or apparatus that allows a user to program, control, evaluate results and/or adjust various aspects of the processing, separation, engineering and formulation steps.
In some aspects, the separation and/or other steps are performed using a clinimmacs system (Miltenyi Biotec), for example, for automatically separating cells at the clinical scale level in closed and sterile systems. The components may include an integrated microcomputer, a magnetic separation unit, peristaltic pumps, and various pinch valves. In some aspects, the integrated computer controls all components of the instrument and directs the system to perform the iterative process in a standardized sequence. In some aspects, the magnetic separation unit includes a movable permanent magnet and a bracket for selecting the column. Peristaltic pumps control the flow rate throughout the tubing set and, in conjunction with pinch valves, ensure that the flow of buffer through the system is controlled and continuous suspension of cells.
In some aspects, the CliniMACS system uses magnetizable particles that provide antibody coupling in a sterile, pyrogen-free solution. In some embodiments, after labeling the cells with magnetic particles, the cells are washed to remove excess particles. The cell preparation bag is then connected to a tube set which in turn is connected to a bag containing buffer and a cell collection bag. The tube set comprises a pre-assembled sterile tube, including a pre-column and a separation column, for single use only. After the separation procedure is initiated, the system automatically applies the cell sample to the separation column. The labeled cells remain in the column, while unlabeled cells are removed by a series of washing steps. In some embodiments, the population of cells used with the methods described herein is unlabeled and not retained in the column. In some embodiments, the population of cells used with the methods described herein is labeled and retained in the column. In some embodiments, after removal of the magnetic field, the population of cells used with the methods described herein is eluted from the column and collected in a cell collection bag.
In certain embodiments, the isolation and/or other steps are performed using a CliniMACS Prodigy system (Miltenyi Biotec). In some aspects, the CliniMACS Prodigy system is equipped with a cell handling unit that allows for automatic washing and fractionation of cells by centrifugation. CliniMACS Prodigy system may also include an on-board camera and image recognition software that determines the optimal cell fractionation endpoint by discriminating the macroscopic layers of the source cell product. For example, peripheral blood automatically separates into red blood cells, white blood cells, and plasma layers. CliniMACS Prodigy systems may also include integrated cell culture chambers that accomplish cell culture protocols such as cell differentiation and expansion, antigen loading, and long term cell culture. The input port may allow sterile removal and replenishment of the culture medium, and the cells may be monitored using an integrated microscope. See, e.g., klebaroff et al (2012) J Immunother.35 (9): 651-660, terakura et al (2012) blood.1:72-82, and Wang et al (2012) J Immunother.35 (9): 689-701.
In some embodiments, the population of cells described herein is collected and enriched (or depleted) by flow cytometry, wherein cells stained for a plurality of cell surface markers are carried in a fluid stream. In some embodiments, the cell populations described herein are collected and enriched (or depleted) by preparative scale (FACS) -sorting. In certain embodiments, the cell populations described herein are collected and enriched (or depleted) by using microelectromechanical systems (MEMS) chips in combination with FACS-based detection systems (see, e.g., WO2010/033140, cho et al (2010) Lab Chip 10,1567-1573; and Godin et al (2008) J Biophoton.1 (5): 355-376. In both cases, cells can be labeled with a variety of markers, allowing well-defined T cell subsets to be isolated in high purity.
In some embodiments, the antibody or binding partner is labeled with one or more detectable markers to facilitate isolation for positive and/or negative selection. For example, the separation may be based on binding to a fluorescently labeled antibody. In some examples, cells are isolated, such as by Fluorescence Activated Cell Sorting (FACS), including, for example, preparation scale (FACS) and/or microelectromechanical systems (MEMS) chips in combination with a flow cell detection system, based on binding of antibodies or other binding partners specific for one or more cell surface markers carried in a fluid flow. Such a method allows positive and negative selection based on multiple markers simultaneously.
In some embodiments, the method of preparation includes the step of freezing, e.g., cryopreserving, the cells prior to or after isolation, incubation, and/or engineering. In some embodiments, the freezing and subsequent thawing steps remove granulocytes and to some extent monocytes from the cell population. In some embodiments, the cells are suspended in a frozen solution, for example followed by a washing step to remove plasma and platelets. In some aspects, any of a variety of known freezing solutions and parameters may be used. One example involves the use of PBS or other suitable cell freezing medium containing 20% dmso and 8% Human Serum Albumin (HSA). Then diluted 1:1 with medium to give final concentrations of DMSO and HSA of 10% and 4%, respectively. The cells were then frozen to-80 ℃ typically at a rate of 1 °/min and stored in the gas phase of a liquid nitrogen storage tank.
In some embodiments, the cells are incubated and/or cultured prior to (or in conjunction with) genetic engineering. The incubation step may include culturing, incubating, stimulating, activating, and/or proliferating. Incubation and/or engineering may be performed in culture vessels such as units, chambers, wells, columns, tubes, tubing sets, valves, vials, petri dishes, bags, or other vessels for culturing or incubating cells. In some embodiments, the composition or cell is incubated in the presence of a stimulating condition or a stimulating agent. Such conditions include those designed to induce proliferation, expansion, activation and/or survival of cells in the population, mimic antigen exposure and/or elicit cells for genetic engineering (such as for the introduction of recombinant antigen receptors).
The conditions may include one or more of the following: specific media, temperature, oxygen content, carbon dioxide content, time, agents, e.g., nutrients, amino acids, antibiotics, ions, and/or stimulatory factors, such as cytokines, chemokines, antigens, binding partners, fusion proteins, recombinant soluble receptors, and any other agents intended to activate cells.
In some embodiments, the stimulating condition or agent comprises one or more agents, e.g., ligands, capable of activating the intracellular signaling domain of the TCR complex. In some aspects, the agent initiates or initiates a TCR/CD3 intracellular signaling cascade in the T cell. Such agents may include antibodies, such as antibodies specific for TCRs, for example anti-CD 3. In some embodiments, the stimulation conditions include one or more agents capable of stimulating a co-stimulatory receptor, such as a ligand, e.g., anti-CD 28. In some embodiments, such agents and/or ligands may be bound to a solid support such as a bead and/or one or more cytokines. Optionally, the amplification method may further comprise the step of adding an anti-CD 3 and/or anti-CD 28 antibody to the culture medium (e.g., at a concentration of at least about 0.5 ng/ml). In some embodiments, the stimulatory agent includes IL-2, IL-15 and/or IL-7. In some aspects, the IL-2 concentration is at least about 10 units/mL.
In some aspects, the incubation is performed according to techniques described, for example, in U.S. Pat. No.6,040,1,77 to Riddell et al, klebaroff et al (2012) J Immunther.35 (9): 651-660, terakura et al (2012) blood.1:72-82, and/or Wang et al (2012) J Immunther.35 (9): 689-701.
In some embodiments, T cells are expanded by adding feeder cells, such as non-dividing Peripheral Blood Mononuclear Cells (PBMCs), to the culture starting composition (e.g., such that the resulting cell population contains at least about 5, 10, 20, or 40 or more PBMC feeder cells for each T lymphocyte in the initial population to be expanded); and incubating the culture (e.g., for a time sufficient to expand the number of T cells). In some aspects, the non-dividing feeder cells can comprise gamma irradiated PBMC feeder cells. In some embodiments, the PBMCs are irradiated with gamma rays in the range of about 3000 to 3600 rads to prevent cell division. In some aspects, feeder cells are added to the medium prior to the addition of the T cell population.
In some embodiments, the stimulation conditions include a temperature suitable for growth of human T lymphocytes, e.g., at least about 25 degrees celsius, typically at least about 30 degrees celsius, and typically at or about 37 degrees celsius. Optionally, the incubating may further comprise adding non-dividing EBV-transformed Lymphoblastoid Cells (LCLs) as feeder cells. The LCL may be irradiated with gamma rays in the range of about 6000 to 10,000 rads. In some aspects, the LCL feeder cells are provided in any suitable amount, such as a ratio of LCL feeder cells to naive T lymphocytes of at least about 10:1.
Composition and method for producing the same
Also provided are compositions, including pharmaceutical compositions and formulations, comprising the binding molecules (e.g., antibodies) provided herein. The compositions and formulations generally comprise one or more optional acceptable carriers or excipients.
The term "pharmaceutical formulation" refers to a formulation which is in a form which allows the biological activity of the active ingredient contained therein to be effective, and which is free of additional components which have unacceptable toxicity to the subject to whom the formulation is administered.
By "pharmaceutically acceptable carrier" is meant an ingredient of the pharmaceutical formulation that is non-toxic to the subject, other than the active ingredient. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, or preservatives.
In some aspects, the choice of vector is determined in part by the particular cell, binding molecule, and/or antibody and/or by the method of administration. Thus, there are a variety of suitable formulations. For example, the pharmaceutical composition may contain a preservative. Suitable preservatives may include, for example, methylparaben, propylparaben, sodium benzoate, and benzalkonium chloride. In some aspects, a mixture of two or more preservatives is used. The preservative or mixtures thereof are typically present in an amount of about 0.0001% to about 2% by weight of the total composition. Vectors are described, for example, in Remington's Pharmaceutical Sciences, 16 th edition, osol, a.ed. (1980). Pharmaceutically acceptable carriers are generally non-toxic to recipients at the dosages and concentrations employed, and include, but are not limited to: buffers such as phosphates, citrates, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (e.g., octadecyldimethylbenzyl ammonium chloride, hexamethyldiammonium chloride, benzalkonium chloride, benzethonium chloride, phenol, butanol, or benzyl alcohol, alkyl parabens such as methyl or propyl parabens, catechol, resorcinol, cyclohexanol, 3-pentanol, and m-cresol); a low molecular weight (less than about 10 residues) polypeptide; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars, such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions, such as sodium; metal complexes (e.g., zn-protein complexes); and/or nonionic surfactants such as polyethylene glycol (PEG).
In some aspects, a buffer is included in the composition. Suitable buffers include, for example, citric acid, sodium citrate, phosphoric acid, potassium phosphate, and various other acids and salts. In some aspects, a mixture of two or more buffers is used. The buffer or mixture thereof is typically present in an amount of about 0.001% to about 4% by weight of the total composition. Methods for preparing administrable pharmaceutical compositions are known. Exemplary methods are described in more detail in, for example, remington, the Science and Practice of Pharmacy, lippincott Williams & Wilkins; 21 st edition (2005, 5 months, 1 day).
In some aspects, the composition may contain a preservative. Suitable preservatives may include, for example, methylparaben, propylparaben, sodium benzoate, and benzalkonium chloride. In some aspects, a mixture of two or more preservatives is used. The preservative or mixtures thereof are typically present in an amount of about 0.0001% to about 2% by weight of the total composition. Vectors are described, for example, in Remington's Pharmaceutical Sciences, 16 th edition, osol, a.ed. (1980). Acceptable carriers include, but are not limited to: buffers such as phosphates, citrates, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (e.g., octadecyldimethylbenzyl ammonium chloride; hexamethyldiammonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butanol or benzyl alcohol; alkyl parabens such as methyl or propyl parabens; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); a low molecular weight (less than about 10 residues) polypeptide; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars, such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions, such as sodium; metal complexes (e.g., zn-protein complexes); and/or nonionic surfactants such as polyethylene glycol (PEG).
Antibody formulations may include lyophilized formulations and aqueous solutions.
In some embodiments, the compositions are provided as sterile liquid formulations, such as isotonic aqueous solutions, suspensions, emulsions, dispersions, or viscous compositions, which in some aspects may be buffered to a selected pH. Liquid formulations are generally easier to prepare than gels, other viscous compositions, and solid compositions. In addition, the liquid composition is somewhat more convenient to administer, in particular by injection. On the other hand, the adhesive composition may be formulated within a suitable viscosity range to provide longer contact times with specific tissues. The liquid or viscous composition may comprise a carrier, which may be a solvent or dispersion medium containing, for example, water, brine, phosphate buffered saline, polyols (e.g., glycerol, propylene glycol, liquid polyethylene glycol), and suitable mixtures thereof.
Sterile injectable solutions may be prepared by incorporating the binding molecules in a solvent, such as a mixture with a suitable carrier, diluent or excipient, such as sterile water, physiological saline, dextrose and the like. The composition may also be lyophilized. The compositions may contain auxiliary substances such as wetting, dispersing or emulsifying agents (e.g., methylcellulose), pH buffering agents, gelling or viscosity-enhancing additives, preservatives, flavouring agents, colouring agents and the like, depending on the route of administration and the desired formulation. In some aspects standard text may be consulted to prepare a suitable formulation.
Various additives that enhance the stability and sterility of the composition may be added, including antimicrobial preservatives, antioxidants, chelating agents, and buffers. Prevention of the action of microorganisms can be ensured by various antibacterial and antifungal agents such as parahydroxybenzoic acid, chlorobutanol, phenol, ascorbic acid and the like. Prolonged absorption of the injectable pharmaceutical form can be brought about by the use of absorption delaying agents, for example, aluminum monostearate and gelatin.
The composition may also be lyophilized. The composition may contain auxiliary substances such as wetting agents, dispersing or emulsifying agents (e.g. methylcellulose), pH buffering agents, gelling or viscosity-enhancing additives, preservatives, colorants and the like. In some aspects standard text may be consulted to prepare a suitable formulation.
Formulations for in vivo administration are generally sterile. Sterility can be readily achieved, for example, by filtration through sterile filtration membranes.
Methods and uses of antibodies
In some embodiments, provided herein are methods involving the use of one or more anti-idiotype antibodies. In some aspects, provided herein are methods for measuring or detecting a target antibody (e.g., CAR or CAR-expressing cell), and methods for modifying target antibody activity (e.g., CAR activity or CAR-expressing cell activity). In certain embodiments, one or more anti-idiotype antibodies bind, detect, identify, and/or quantify the CAR and/or CAR-expressing cells. In some embodiments, the methods provided herein provide one or more steps of contacting and/or incubating one or more anti-idiotype antibodies with a cell expressing a Chimeric Antigen Receptor (CAR) or a sample containing or believed to contain the cell. In some embodiments, the anti-idiotype antibody is treated, incubated, and/or contacted with the composition or sample under conditions that allow a complex to form between the anti-idiotype antibody and the target antibody (e.g., CAR). In some aspects, the complexes can be used for the purpose of detecting, isolating, and/or measuring CARs. In some embodiments, the formation of the complex alters the activity of the target antibody (e.g., CAR), such as by stimulating receptor signaling activity, or in some embodiments, antagonizing the activity of the target antibody (e.g., CAR) by preventing the CAR from binding to an antigen.
A. Detection/separation method
In some embodiments, methods are provided that involve the detection, binding and/or isolation of antibodies (e.g., target antibodies) using one or more anti-idiotype antibodies and/or molecules (such as conjugates and complexes) containing one or more such anti-idiotype antibodies. In certain embodiments, the methods provide one or more steps of contacting, incubating, and/or exposing one or more anti-idiotype antibodies with a sample and/or composition. In some embodiments, the sample and/or composition has, may have, and/or is suspected of having, a target antibody and/or antigen-binding fragment thereof that is bound and/or recognized by one or more anti-idiotype antibodies. In certain embodiments, an antibody or antigen-binding fragment thereof that is bound or recognized by one or more anti-idiotype antibodies contains one or more fusion domains and/or is a fusion protein. In certain embodiments, the target antibody or antigen binding fragment thereof is a CAR. In certain embodiments, the anti-idiotype antibody binds to and/or recognizes an anti-CD 19 antibody (e.g., antibody SJ25C1 or FMC 63) or antigen binding fragment thereof, including chimeric molecules or conjugates comprising a CAR containing such an anti-CD 19 antibody (e.g., antibody fragment).
Methods in some embodiments include incubating, treating, and/or contacting a sample and/or composition containing or suspected of containing a target antibody with an anti-idiotype antibody. In certain embodiments, the incubation is under conditions that allow the anti-idiotype antibody to bind to the target antibody present in the composition, e.g., so as to form a complex containing the anti-idiotype antibody and the target antibody.
In some embodiments, the sample and/or composition contains or is suspected of containing a target antibody, e.g., a CAR. In certain embodiments, the sample and/or composition contains or is suspected of containing cells expressing a target antibody, e.g., a CAR. In certain embodiments, the sample is a biological sample. In particular embodiments, the sample is a serum sample and/or a blood sample. In some embodiments, the biological sample contains one or more immune cells. In some embodiments, the biological sample is or is derived from tissue, such as connective tissue, muscle tissue, nerve tissue, or epithelial tissue. In particular embodiments, the biological sample is or is derived from the heart, vasculature, salivary gland, esophagus, stomach, liver, gall bladder, pancreas, intestine, colon, rectum, hypothalamus, pituitary gland, pineal gland, thyroid gland, parathyroid gland, adrenal gland, kidney, ureter, bladder, urinary tract, lymphatic system, skin, muscle, brain, spinal cord, nerve, ovary, uterus, testis, prostate, pharynx, larynx, trachea, bronchi, lung, diaphragm, bone, cartilage, ligament or tendon. In particular embodiments, a biological sample is taken, collected and/or obtained from a human subject. In certain embodiments, the sample contains living cells and/or intact cells. In some embodiments, the sample is or contains homogenates and/or cells that have been destroyed and/or lysed. In some embodiments, the biological sample contains proteins and/or antibodies that have been isolated from blood, serum, and/or tissue.
In particular embodiments, the anti-idiotype antibody forms or is capable of forming a complex with a target antibody (e.g., CAR). In particular embodiments, the complex is detected, measured, quantified, and/or assessed, e.g., to allow detection, identification, measurement, and/or quantification of, e.g., a target antibody in a composition or sample. In certain embodiments, the methods comprise detecting whether a complex is formed between the anti-idiotype antibody and the target antibody in the sample, and/or detecting the presence or absence or level of such binding. In some embodiments, the complex contains a detectable label. In particular embodiments, the anti-idiotype antibody is an immunoconjugate comprising a detectable label. In certain embodiments, the anti-idiotype antibody contains, is conjugated to, is bound to, and/or is attached to a detectable label. In some embodiments, the complex contains an antibody, e.g., a secondary antibody, that binds to and/or recognizes an anti-idiotype antibody, conjugated to, bound to, and/or attached to a detectable label.
In some embodiments, a method for detecting, quantifying, detecting, and/or assessing a target antibody in, for example, a sample or composition, comprises detecting a complex of the target antibody and an anti-idiotype antibody. In some embodiments, the complex contains a detectable label. In certain embodiments, the complex is detected and/or contacted with a detectable label. In some embodiments, the complex is detected by any suitable method or means, such as, but not limited to, flow cytometry, immunocytochemistry, immunohistochemistry, western blot analysis, and ELISA.
In some embodiments, the target antibody or antigen binding fragment binds to or is expressed on the surface of a cell. In particular embodiments, the target antibody (e.g., CAR) does not bind to or is not contained within a cell, e.g., in some embodiments, the target antibody is secreted. In certain embodiments, the antibody has been isolated, removed, and/or lysed from the cell surface.
In some embodiments, the target antibody or antigen binding fragment is comprised in a Chimeric Antigen Receptor (CAR), such as a CAR expressed on the surface of a cell. In some embodiments, the cell is a stem cell (e.g., iPSC) or an immune cell. In some embodiments, the immune cell is a T cell, e.g., a cd4+ T cell, a cd8+ T cell, a naive T (T) N ) Cells, effector T cells (T EFF ) Memory T cells, tumor Infiltrating Lymphocytes (TILs), immature T cells, mature T cells, helper T cells, cytotoxic T cells, mucosa-associated invariant T (MAIT) cells, naturally occurring and adapted regulatory T (Treg) cells, helper T cells, such as TH1 cells, TH2 cells, TH3 cells, TH17 cells, TH9 cells, TH22 cells, follicular helper T cells, alpha/beta T cells, and/or delta/gamma T cells. In some embodiments, the cells are from a tissue, such as heart, vasculature, salivary gland, esophagus, stomach, liver, gall bladder, pancreas, intestine, colon, rectum, hypothalamus, pituitary gland, pineal gland, thyroid gland, parathyroid gland Adrenal gland, kidney, ureter, bladder, urethra, lymphatic system, skin, muscle, brain, spinal cord, nerve, ovary, uterus, testis, prostate, pharynx, larynx, trachea, bronchi, lung, diaphragm, bone, cartilage, ligament or tendon.
In some embodiments, the target antibody is an anti-CD 19 antibody. In some embodiments, the target antibody is or is derived from antibody SJ25C1 or an antigen binding fragment thereof. In some embodiments, the target antibody or antigen-binding fragment thereof comprises the heavy chain variable region set forth in SEQ ID NO. 23 and/or the light chain variable region set forth in SEQ ID NO. 24. In some embodiments, the target antibody is or is derived from antibody FMC63 or an antigen binding fragment thereof. In some embodiments, the target antibody or antigen-binding fragment thereof comprises the heavy chain variable region set forth in SEQ ID NO. 30 and/or the light chain variable region set forth in SEQ ID NO. 31.
In some embodiments, methods of detecting a target antibody, such as antibody SJ25C1 or FMC63, or an antigen binding fragment thereof (and/or a chimeric molecule comprising such an antibody, e.g., an antibody fragment, such as a CAR) are provided, the methods comprising contacting a composition comprising the target antibody or antigen binding fragment with an anti-idiotype antibody or antigen binding fragment thereof or an anti-idiotype antibody immunoconjugate described herein, and detecting an anti-idiotype antibody that binds to the target antibody or antigen binding fragment. In some embodiments, the method further comprises detecting whether a complex is formed between the anti-idiotype antibody and the target antibody in the composition, such as detecting the presence or absence or level of such binding. In some embodiments, the target antibody or antigen binding fragment binds to or is expressed on the surface of a cell, and the detecting comprises detecting a cell that binds to the anti-idiotype antibody. In some embodiments, an anti-idiotype antibody or antigen-binding fragment thereof is labeled, directly or indirectly, for detection. In some embodiments, the target antibody or antigen-binding fragment thereof comprises the heavy chain variable region set forth in SEQ ID NO. 23 and/or the light chain variable region set forth in SEQ ID NO. 24. In some embodiments, the target antibody or antigen-binding fragment thereof comprises the heavy chain variable region set forth in SEQ ID NO. 30 and/or the light chain variable region set forth in SEQ ID NO. 31.
In some embodiments, methods of isolating a target antibody, such as antibody SJ25C1 or FMC63, or an antigen binding fragment thereof (and/or chimeric molecules comprising such an antibody, e.g., an antibody fragment, such as a CAR) are provided, comprising contacting a composition and/or sample containing or suspected of containing the target antibody or antigen binding fragment with an anti-idiotype antibody, or antigen binding fragment thereof, or an anti-idiotype antibody immunoconjugate described herein, and isolating a complex comprising the anti-idiotype antibody bound to the target antibody or antigen binding fragment. In certain embodiments, the target antibody is isolated with an anti-idiotype antibody or antigen-binding fragment thereof (as an immunoconjugate), such as the immunoconjugate described in sections I-F. In some embodiments, the target antibody or antigen binding fragment binds to or is expressed on the surface of a cell, and the isolating comprises isolating the cell that binds to the anti-idiotype antibody. In some embodiments, the complex comprising the anti-idiotype antibody is separated by affinity-based separation. In some embodiments, the affinity-based separation is selected from the group consisting of: immunoaffinity-based separations, magnetic-based separations, and affinity chromatography. In some embodiments, the target antibody or antigen-binding fragment thereof comprises the heavy chain variable region set forth in SEQ ID NO. 23 and/or the light chain variable region set forth in SEQ ID NO. 24. In some embodiments, the target antibody or antigen-binding fragment thereof comprises the heavy chain variable region set forth in SEQ ID NO. 30 and/or the light chain variable region set forth in SEQ ID NO. 31.
In some embodiments, methods of detecting a cell expressing a CAR comprising a target antibody, such as antibody SJ25C1 or FMC63, or an antigen binding fragment thereof, are provided, the methods comprising contacting the cell expressing the CAR with an anti-idiotype antibody or antigen binding fragment thereof or an anti-idiotype antibody immunoconjugate described herein, and detecting a cell that binds to the anti-idiotype antibody. In some embodiments, an anti-idiotype antibody or antigen-binding fragment thereof is labeled, directly or indirectly, for detection. In some embodiments, the target antibody or antigen-binding fragment thereof comprises the heavy chain variable region set forth in SEQ ID NO. 23 and/or the light chain variable region set forth in SEQ ID NO. 24. In some embodiments, the target antibody or antigen-binding fragment thereof comprises the heavy chain variable region set forth in SEQ ID NO. 30 and/or the light chain variable region set forth in SEQ ID NO. 31.
In certain embodiments, the methods of detecting a target antibody with an anti-idiotype antibody described herein are used to evaluate the target antibody in a subject. For example, in some embodiments, provided herein are methods of use of an anti-idiotype antibody for assessing, measuring, and/or quantifying in vivo pharmacokinetics, expansion, and/or persistence of a CAR-expressing cell of a therapeutic cell composition. In some embodiments, changes in vivo pharmacokinetics, expansion and/or persistence, and/or cellular phenotype or functional activity of cells, such as CAR-expressing cells administered by immunotherapy (e.g., CAR-T cell therapy), in the methods provided herein can be measured with the anti-idiotype antibodies provided herein. In some embodiments, the pharmacokinetics, expansion, and/or persistence of the CAR-expressing cells are assessed, measured during and/or after administration of the therapy by detecting the presence and/or amount of CAR-expressing cells in a subject and/or in a sample obtained from the subject with an anti-idiotype antibody provided herein after administration of a therapeutic cell composition.
In some aspects, the anti-idiotype antibody is used in conjunction with flow cytometry to assess the number of cells expressing a recombinant receptor (e.g., CAR-expressing cells administered for T-cell based therapy) in a blood or serum or organ or tissue sample (e.g., a disease site, such as a tumor sample) of a subject. In some aspects, persistence is quantified as the number of CAR-expressing cells per microliter of sample (e.g., blood or serum) or as a total number of Peripheral Blood Mononuclear Cells (PBMCs) or leukocytes or T cells per microliter of sample. In certain aspects, the expansion is quantified as an increase in the number of CAR-expressing cells per microliter of sample (e.g., blood or serum) or in terms of total number of Peripheral Blood Mononuclear Cells (PBMCs) or leukocytes or T cells per microliter of sample over time. In some embodiments, the pharmacokinetics, amplification, and/or persistence are measured or assessed by detecting the amount of CAR-expressing cells in a subject at a plurality of time points and/or in a sample collected from the subject. In certain embodiments, one or more samples are collected, obtained, and/or taken from a subject 24 hours, 48 hours, 72 hours, 4 days, 5 days, 6 days, 7 days, 10 days, 14 days, 21 days, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, 3 months, 4 months, 6 months, within one year or more after administration of the therapeutic cell composition.
In some embodiments, methods of selecting cells expressing a CAR comprising a target antibody, such as antibody SJ25C1 or FMC63, or antigen binding fragment thereof, are provided, comprising contacting a population of cells comprising cells expressing a CAR with an anti-idiotype antibody or antigen binding fragment thereof or an anti-idiotype antibody immunoconjugate described herein, and selecting cells that bind to the anti-idiotype antibody. In some embodiments, cells that bind to the anti-idiotype antibody are selected by affinity-based separation. In some embodiments, the affinity-based separation is selected from the group consisting of: immunoaffinity-based separations, flow cytometry, magnetic-based separations, and affinity chromatography. In some embodiments, the anti-idiotype antibody or antigen-binding fragment thereof or anti-idiotype antibody immunoconjugate is reversibly bound or immobilized on a support or stationary phase. In some embodiments, the target antibody or antigen-binding fragment thereof comprises the heavy chain variable region set forth in SEQ ID NO. 23 and/or the light chain variable region set forth in SEQ ID NO. 24. In some embodiments, the target antibody or antigen-binding fragment thereof comprises the heavy chain variable region set forth in SEQ ID NO. 30 and/or the light chain variable region set forth in SEQ ID NO. 31.
In some embodiments, a method of validating a CAR comprising a target antibody, such as antibody SJ25C1 or FMC63, or an antigen binding fragment thereof, is provided, the method comprising a) incubating a sample comprising T cells transduced with the CAR with an anti-idiotype antibody or antigen binding fragment thereof that targets the CAR; b) Determining the percentage of cells bound to the anti-idiotype antibody or antigen-binding fragment thereof; and c) validating the CAR based on the percentage of T cells bound by the anti-idiotype antibody. In some embodiments, the anti-idiotype antibody is labeled and the T cells to which the anti-idiotype antibody binds are determined by flow cytometry. In some embodiments, the target antibody or antigen-binding fragment thereof comprises the heavy chain variable region set forth in SEQ ID NO. 23 and/or the light chain variable region set forth in SEQ ID NO. 24. In some embodiments, the target antibody or antigen-binding fragment thereof comprises the heavy chain variable region set forth in SEQ ID NO. 30 and/or the light chain variable region set forth in SEQ ID NO. 31.
Also provided are methods involving the use of provided anti-idiotype antibodies and molecules (such as conjugates and complexes) containing one or more such anti-idiotype antibodies for signaling a therapeutic decision to an individual, such as by detecting a CAR recognized by the anti-idiotype antibody, such as a CAR comprising a target antibody, such as an anti-CD 19 antibody (e.g., antibody SJ25C1 or FMC 63) or an antigen binding fragment thereof. In some embodiments, the methods are used to inform an individual of a treatment decision associated with a therapy comprising administration of CAR T cells (e.g., anti-CD 19CAR T cells). In some embodiments, the methods comprise incubating and/or detecting a biological sample with an anti-idiotype antibody and/or administering an anti-idiotype antibody to an individual. In certain embodiments, the biological sample comprises cells or tissue or a portion thereof, such as a tumor or cancer tissue or biopsy or a section thereof. In certain embodiments, the incubation is under conditions that allow the anti-idiotype antibody to bind to a CAR present in the sample. In some embodiments, the method further comprises detecting whether a complex is formed between the anti-idiotype antibody and the CAR in the sample, such as detecting the presence or absence or level of such binding. Such methods may be in vitro or in vivo.
In one embodiment, the anti-idiotype antibody is used to determine whether modulation of CAR T cell therapy in an individual is required, e.g., wherein a low level of CAR T cells in the individual indicates that modulation of the therapy is required. In some embodiments, the target antibody is an anti-CD 19 antibody. In some embodiments, the target antibody is or is derived from antibody SJ25C1 or an antigen binding fragment thereof. In some embodiments, the target antibody or antigen-binding fragment thereof comprises the heavy chain variable region set forth in SEQ ID NO. 23 and/or the light chain variable region set forth in SEQ ID NO. 24. In some embodiments, the target antibody is or is derived from antibody FMC63 or an antigen binding fragment thereof. In some embodiments, the target antibody or antigen-binding fragment thereof comprises the heavy chain variable region set forth in SEQ ID NO. 30 and/or the light chain variable region set forth in SEQ ID NO. 31.
In some embodiments, a method of assessing CAR T cell therapy in an individual is provided, wherein the CAR comprises a target antibody, such as antibody SJ25C1 or FMC63, or an antigen binding fragment thereof, the method comprising incubating a sample from the individual with an anti-idiotype antibody or antigen binding fragment thereof that targets the CAR, and determining the amount of T cells bound to the anti-idiotype antibody, and determining the potential therapeutic benefit of the therapy based on the amount of T cells bound to the anti-idiotype antibody. In some embodiments, the anti-idiotype antibody is labeled and the T cells to which the anti-idiotype antibody binds are determined by flow cytometry. In some embodiments, the sample is a blood-derived sample, or is derived from a blood component apheresis or a leukocyte apheresis product. In some embodiments, the target antibody or antigen-binding fragment thereof comprises the heavy chain variable region set forth in SEQ ID NO. 23 and/or the light chain variable region set forth in SEQ ID NO. 24. In some embodiments, the target antibody or antigen-binding fragment thereof comprises the heavy chain variable region set forth in SEQ ID NO. 30 and/or the light chain variable region set forth in SEQ ID NO. 31.
In some embodiments, methods of assessing CAR T cell therapy in an individual are provided. In some aspects, the CAR comprises a target antibody, such as an antibody that is or is derived from SJ25C1 or FMC63, such as an antigen binding fragment of full length SJ25C1 or FMC 63. In some embodiments, the method comprises administering to the individual an anti-idiotype antibody or antigen-binding fragment thereof that targets the CAR (e.g., an antibody that targets the CAR, e.g., an antibody fragment). In some aspects, administration is performed after the first dose of the therapy is initiated. The method may comprise determining the presence of anti-idiotype antibodies in one or more tissues/organs in the individual. In some aspects, the method comprises determining a potential therapeutic benefit of the therapy based on the presence of an anti-idiotype antibody in at least one of the one or more tissues/organs. In some embodiments, the anti-idiotype antibody is labeled, and the presence of the anti-idiotype antibody in the individual is determined by imaging in the individual to detect the label. In some embodiments, determining the presence of anti-idiotype antibodies in one or more tissues/organs in the individual comprises determining the level of anti-idiotype antibodies in one or more tissues/organs, or a combination thereof, or is performed by said determining.
In some embodiments, the method further comprises administering an anti-idiotype antibody to the individual after a second or subsequent dose of the therapy is initiated and/or determining the presence of the anti-idiotype antibody in one or more tissues/organs in the individual. In some aspects, the method further involves determining a potential therapeutic benefit of the therapy based on a difference in anti-idiotype antibody levels in at least one of the one or more tissues/organs in the individual between the first and second anti-idiotype antibody administrations. In some embodiments, the target antibody or antigen-binding fragment thereof comprises the heavy chain variable region set forth in SEQ ID NO. 23 and/or the light chain variable region set forth in SEQ ID NO. 24. In some embodiments, the target antibody or antigen-binding fragment thereof comprises the heavy chain variable region set forth in SEQ ID NO. 30 and/or the light chain variable region set forth in SEQ ID NO. 31.
In some embodiments, methods of modulating CAR T cell therapy in an individual are provided, wherein the CAR comprises a target antibody, such as antibody SJ25C1 or FMC63, or an antigen binding fragment thereof. In some aspects, the method comprises incubating or contacting a sample from the individual with an anti-idiotype antibody or antigen-binding fragment thereof that targets the CAR. In some aspects, the methods comprise determining the amount of T cells bound to or associated with an anti-idiotype antibody. In some aspects, the methods comprise modulating the therapy based on the amount of T cells bound by the anti-idiotype antibody. In some embodiments, the anti-idiotype antibody is labeled directly or indirectly. In some aspects of such embodiments, for example, in some cases, the anti-idiotype antibody-bound T cells are imaged in vivo or ex vivo by flow cytometry by assaying a sample from a subject administered the CAR-T cells and the anti-idiotype antibody. In some embodiments, the sample is a blood-derived sample and/or is derived from a blood component apheresis or a leukocyte apheresis product. In some embodiments, the target antibody or antigen-binding fragment thereof comprises the heavy chain variable region set forth in SEQ ID NO. 23 and/or the light chain variable region set forth in SEQ ID NO. 24. In some embodiments, the target antibody or antigen-binding fragment thereof comprises the heavy chain variable region set forth in SEQ ID NO. 30 and/or the light chain variable region set forth in SEQ ID NO. 31.
In some embodiments, methods of modulating CAR T cell therapy in an individual are provided. In some aspects, such methods are practiced on CAR-T cell therapies, wherein the CAR comprises a target antibody, such as antibody SJ25C1 or FMC63, including antigen binding fragments of SJ25C1 or FMC 63. In some embodiments, the method comprises administering to the individual an anti-idiotype antibody or antigen-binding fragment thereof that targets or binds to the CAR after starting the first dose of the therapy and determining the presence, absence, or level of the anti-idiotype antibody in one or more tissues/organs in the individual. In some aspects, the methods comprise modulating the therapy based on the presence, absence, or level of anti-idiotype antibodies in at least one of the one or more tissues/organs. In some embodiments, the anti-idiotype antibody is labeled directly or indirectly, and in some such embodiments the presence of the anti-idiotype antibody in the individual is determined by imaging in the individual to detect the label. In some embodiments, determining the presence of anti-idiotype antibodies in one or more tissues/organs in the individual comprises determining the level of anti-idiotype antibodies in one or more tissues/organs or binding thereof. In some embodiments, the method further comprises administering an anti-idiotype antibody to the subject after a second or subsequent dose of the therapy is initiated, and in some aspects, determining the presence, absence, or level of anti-idiotype antibody in one or more tissues/organs in the subject, and/or adjusting the therapy based on observations (such as based on differences in anti-idiotype antibody levels in at least one of the one or more tissues/organs in the subject between the first and second anti-idiotype antibody administrations). In some embodiments, the target antibody or antigen-binding fragment thereof comprises the heavy chain variable region set forth in SEQ ID NO. 23 and/or the light chain variable region set forth in SEQ ID NO. 24. In some embodiments, the target antibody or antigen-binding fragment thereof comprises the heavy chain variable region set forth in SEQ ID NO. 30 and/or the light chain variable region set forth in SEQ ID NO. 31.
In some aspects, the therapy is adjusted in the following cases: (i) If the number of cells of the T cell therapy detectable in the blood or other biological sample after administration of the T cell therapy is undetectable or reduced after having been detectable (optionally reduced compared to the previous time point); (ii) After the initiation of administration of the T cell therapy (optionally the first, second, or subsequent dose), the number of cells of the detectable T cell therapy is reduced (or reduced by more than) a 1.5-fold, 2.0-fold, 3.0-fold, 4.0-fold, 5.0-fold, 10-fold, or more in the blood or other biological sample of the subject compared to the peak or maximum number of cells of the detectable T cell therapy in the blood or biological sample of the subject; (iii) At a time after a peak or maximum level of cells of the T cell therapy is detectable in the blood of the subject, less than 10%, less than 5%, less than 1%, or less than 0.1% of the total Peripheral Blood Mononuclear Cells (PBMCs) in the blood of the subject of the detectable T cells in the blood of the subject or cells derived therefrom; and/or (iv) if the number of cd3+ or cd8+ cells of the cell therapy detectable in the blood is less than 20 cells/μl, 15 cells/μl, 10 cells/μl, less than 5 cells/μl or less than 1 cell/μl. In some embodiments, the therapy is adjusted by: administering one or more additional doses of the CAR-T cell therapy, administering an increased dose of the CAR-T cell therapy, administering an alternative CAR-T cell therapy specific for the same or a different antigen, administering one or more immunomodulators or other agents that promote or increase the expansion or persistence of CAR-T cells.
Various methods known in the art for detecting specific antibody-antigen binding may be used. Showing theExemplary immunoassays include Fluorescence Polarization Immunoassay (FPIA), fluorescence Immunoassay (FIA), enzyme Immunoassay (EIA), turbidimetric inhibition immunoassay (NIA), enzyme-linked immunosorbent assay (ELISA), and Radioimmunoassay (RIA). An indicator moiety or marker group may be attached to the anti-idiotype antibody and selected to meet the needs of the various uses of the method, as generally determined by the availability of the assay device and the compatible immunoassay procedure. Exemplary labels include radionuclides (e.g 125 I、 131 I、 35 S、 3 H or 32 P and/or Cr 51 Cr, co% 57 Co and F 18 F) Gadolinium% 153 Gd、 159 Gd, germanium% 68 Ge, holmium 166 Ho, indium% 115 In、 113 In、 112 In、 111 In and iodine% 125 I、 123 I、 121 I) Lanthanum% 140 La, lutetium ] 177 Lu and Mn 54 Mn, mo 99 Mo and Pd 103 Pd and P ] 32 P, praseodymium% 142 Pr and promethium 149 Pm, re ] 186 Re、 188 Re and rhodium% 105 Rh), ruthenium (ruthenium) 97 Ru and samarium 153 Sm, scandium 47 Sc and Se% 75 Se)、( 85 Sr and S 35 S), technetium 99 Tc), thallium 201 Ti, sn 113 Sn、 117 Sn, tritium 3 H) Xe 133 Xe, ytterbium% 169 Yb、 175 Yb and yttrium% 90 Y); enzymes (e.g., alkaline phosphatase, horseradish peroxidase, luciferase, or beta-galactosidase); fluorescent modules or proteins (e.g., fluorescein, rhodamine, phycoerythrin, GFP, or BFP); or a light emitting module (e.g., qdot provided by Quantum Dot Corporation, palo Alto, calif.) TM Nanoparticles). Various general techniques for performing the various immunoassays described above are known.
In some embodiments, it is not necessary to label the anti-idiotype antibody, and the presence thereof can be detected using a labeled antibody that binds to any anti-idiotype antibody.
The anti-idiotype antibodies provided herein can be used in any known assay, such as competitive binding assays, direct and indirect sandwich assays, and immunoprecipitation assays. Zola, monoclonal Antibodies: A Manual of Techniques, pp.147-158 (CRC Press, inc. 1987).
Anti-idiotype antibodies may also be used in vivo diagnostic assays, such as in vivo imaging. Typically, anti-idiotype antibodies are conjugated with radionuclides (e.g 111 In、 99 Tc、 14 C、 131 I、 125 I or 3 H) Markers such that cells or tissues of interest can be located in vivo after administration to an individual.
In some embodiments, the anti-idiotype antibody or antigen-binding fragment is immobilized or bound to a solid support, wherein one or more target cells, including CAR-T cells, are contacted with the solid support. In some embodiments, the solid support is a bead. In some embodiments, the solid support is a surface of a well or plate (e.g., a cell culture plate). In some embodiments, the solid support is a resin or matrix present in or contained within the chromatography column, e.g., to allow chromatographic separation or selection of car+ T cells. In some embodiments, the anti-idiotype antibody or antigen-binding fragment is reversibly or can be reversibly bound to a solid support. In some embodiments, the solid support is an affinity chromatography matrix comprising one or more binding sites capable of binding (e.g., reversibly binding) to a binding partner present in the anti-idiotype antibody. In one exemplary embodiment, the anti-idiotype antibody comprises a streptavidin binding peptide or other streptavidin binding moiety that is capable of binding to a streptavidin or streptavidin mutein molecule present or immobilized on a solid support, in some cases, that can be dissociated in the presence of a competing substance such as biotin. Examples of such systems include those described in U.S. published patent application number US 20150024411.
In some aspects, the anti-ID antibodies provided herein can be expressed on the surface of a cell. In some aspects, the cell-expressed anti-ID antibodies can be used to induce or stimulate CAR-expressing cells, such as part of a system for selectively growing CAR cells. In some aspects, the anti-ID antibody or antigen-binding fragment thereof is expressed on an artificial antigen presenting cell (aAPC). Aapcs expressing anti-ID can be used as agents to stimulate or expand CAR T cell populations.
Methods for preparing or generating aapcs are known, see for example U.S. Pat. nos. 6,225,042, 6,355,479, 6,362,001, 6,790,662, 7,754,482; U.S. patent application publication nos. 2009/0017000 and 2009/0004142; and International publication No. WO2007/103009. Various aapcs are known in the art, see for example U.S. patent No.8,722,400, published application number US2014/0212446; butler and Hirano (2014) Immunol rev 257 (1): 10.1111/imr.12129; suhoshki et al (2007) mol. Ther., 15:981-988).
aapcs include features of natural APCs, including MHC molecules, stimulatory and co-stimulatory molecules, fc receptors, adhesion molecules, and/or the ability to produce or secrete cytokines (e.g., IL-2). Generally, aapcs are cell lines that lack expression of one or more of the above molecules and are generated by introducing (e.g., by transfection or transduction) one or more deletion elements necessary to stimulate a cell (e.g., a CAR-T cell).
In some embodiments, the cells selected to become aapcs have defects in intracellular antigen processing, intracellular peptide trafficking, and/or intracellular MHC class I or class II molecule-peptide loading. In some aspects, such cells also lack the ability to express MHC class I or class II molecules and/or molecules involved in or involved in antigen processing. Exemplary aapcs constitute or are derived from a transporter associated with an antigen processing (TAP) defective cell line, such as an insect cell line. An exemplary cell line is a Drosophila (Drosophila) cell line, such as the Schneider 2 cell line (see, e.g., schneider, J. Embryol. Exp. Morph.1972Vol 27, pp. 353-365). Illustrative methods for the preparation, growth and culture of Schneider 2 cells are provided in U.S. Pat. Nos. 6,225,042, 6,355,479 and 6,362,001.
In some embodiments, the cell is a K652 cell or a K562 derived cell. In some embodiments, the cell is a cell line obtainable at ATCC No. CCL-243.
In some aspects, the aapcs are further engineered to express additional molecules to enhance, boost, or augment stimulation of CAR-expressing T cells. In some embodiments, the additional molecule is an immunostimulatory ligand, a co-stimulatory ligand, a cytokine or an adhesion molecule. In some embodiments, the costimulatory ligand specifically binds to at least one costimulatory molecule present on the T cell. In some embodiments, aapcs are produced, e.g., by transduction or transfection, to express one or more costimulatory signals (e.g., antibodies that specifically bind to a ligand of CD7, B7-1 (CD 80), B7-2 (CD 86), PD-L1, PD-L2, 4-1BBL, OX40L, ICOS-L, ICAM, CD30L, CD, CD70, CD83, HLA-G, MICA, MICB, HVEM, lymphotoxin β receptor, ILT3, ILT4, 3/TR6, or B7-H3; or to a ligand of CD27, CD28, 4-1BB, OX40, CD30, CD40, PD-1, ICOS, LFA-1, CD2, CD7, LIGHT, NKG2C, B-H3, toll ligand receptor, or CD 83), cell adhesion molecules (e.g., ICAM-1 or LFA-3), and/or cytokines (e.g., IL-2, IL-4, IL-6, IL-7, IL-10, IL-12, IL-21, IL- β), tumor necrosis factor (TNF- α), tumor necrosis factor (GM- α), tumor factor (TNF- α), and the colony- α (α)). In some cases, aapcs do not normally express MHC molecules, but can be engineered to express MHC molecules, or in some cases, can be induced or inducible to express MHC molecules, such as by stimulation with cytokines. In some cases, aapcs may also be loaded with stimulating or co-stimulating ligands, which may include, for example, anti-CD 3 antibodies, anti-CD 28 antibodies, or anti-CD 2 antibodies. In some embodiments, aapcs express molecules capable of mediating a primary signal in a cell, such as through a T cell receptor/CD 3 complex on a T cell. In some embodiments, aapcs comprise a stimulatory ligand that specifically binds to the TCR/CD3 complex, thereby transducing a primary signal.
In some embodiments, aapcs do not express a stimulatory ligand that specifically binds to the TCR/CD3 complex, as the anti-ID is capable of delivering a signal through the CAR. In some embodiments, aapcs do not express co-stimulatory molecules.
In some embodiments, the anti-ID is expressed as a single chain fragment (scFv) expressed on the cell surface. Nucleic acids encoding scfvs can be fused to DNA sequences encoding transmembrane domains. Specific uses of the transmembrane region include those derived from the alpha, beta or zeta chain of a T cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD 154. Alternatively, the transmembrane domain may be synthetic, in which case it will predominantly comprise hydrophobic residues such as leucine and valine. In some cases, triplets of phenylalanine, tryptophan and valine will be found at each end of the synthetic transmembrane domain. Exemplary transmembrane domains include those derived from CD8 or CD 28.
B. Use in cell stimulation
In some embodiments, the provided anti-idiotype antibodies or antigen-binding fragments thereof are agonists and/or exhibit specific activity to stimulate cells expressing a target antibody, including conjugates or chimeric receptors containing the same, such as an anti-CD 19 antibody (e.g., antibody SJ25C1 or FMC 63) or antigen-binding fragment thereof. In some embodiments, methods are provided that involve the use of the provided anti-idiotype antibodies and molecules (such as conjugates and complexes) containing one or more such anti-idiotype antibodies for stimulating or activating cells expressing a CAR or cells expressing other chimeric receptors (such as T cells). In some aspects, the CAR or other receptor comprises a target antibody, such as an anti-CD 19 antibody (e.g., antibody SJ25C1 or FMC 63) or an antigen binding fragment thereof.
In some embodiments, the methods can be used in conjunction with methods of making genetically engineered T cells, such as methods for expanding genetically engineered T cells or other cells in which a nucleic acid molecule encoding a chimeric receptor (e.g., CAR) comprising a target antibody has been introduced, for example, by transfection, transduction, or nucleic acid transfer, in a non-viral manner (e.g., transposon based methods). In some aspects, the target antibody is an anti-CD 19 antibody (e.g., antibody SJ25C1 or FMC 63) or an antigen binding fragment thereof. In particular embodiments, the target antibody is or contains a CAR, e.g., an anti-CD 19CAR. In particular embodiments, the anti-CD 19CAR contains an scFv derived from and/or derived from an anti-CD 19 antibody, such as antibody SJ25C1 or FMC63.
The method in some embodiments comprises incubating a sample comprising T cells transduced with the CAR with an anti-idiotype antibody. In certain embodiments, the method further comprises detecting whether the CAR T cell is activated or stimulated, such as by assessing the viability, proliferation, and/or expression of an activation marker in the CAR T cell. In some embodiments, the target antibody is an anti-CD 19 antibody. In some embodiments, the target antibody is or is derived from antibody SJ25C1 or FMC63 or antigen binding fragment thereof. In some embodiments, the target antibody or antigen-binding fragment thereof comprises the heavy chain variable region set forth in SEQ ID NO. 23 and/or the light chain variable region set forth in SEQ ID NO. 24. In some embodiments, the target antibody or antigen-binding fragment thereof comprises the heavy chain variable region set forth in SEQ ID NO. 30 and/or the light chain variable region set forth in SEQ ID NO. 31.
In some embodiments, methods of mimicking cells are provided, comprising incubating an input composition comprising cells expressing a CAR (comprising a target antibody, such as antibody SJ25C1 or FMC63, or an antigen binding fragment thereof) with an anti-idiotype antibody or antigen binding fragment thereof described herein, thereby generating an output composition comprising stimulated cells. In some embodiments, the incubation is performed under conditions in which the anti-idiotype antibody or antigen-binding fragment thereof binds to the CAR, thereby inducing or modulating a signal in one or more cells of the input composition. In some embodiments, the cells comprise T cells. In some embodiments, the T cells comprise cd4+ and/or cd8+ T cells.
In some embodiments, provided herein are methods of stimulating or amplifying a cell expressing a CAR by incubating an input composition containing the cell expressing the CAR with an anti-idiotype antibody that binds and/or recognizes the CAR. In some embodiments, the binding between the anti-idiotype antibody and the CAR induces expansion of the CAR-expressing cell, thereby producing an output composition comprising the expanded cell.
In some embodiments, the anti-idiotype antibody is contacted or incubated with an input composition having one or more cells to produce an output composition. In certain embodiments, the input cell and/or input composition is a composition and/or a plurality of cells that are (or are expected to be) treated, incubated or contacted under conditions that will produce one or more changes to at least a portion of the cells of the input composition, thereby converting the input composition into an output composition. In some embodiments, the input cell is a composition of immune cells, e.g., a composition of T cells containing cells expressing the CAR. In particular embodiments, by practicing the provided methods, at least a portion of the cells in the input composition are activated, expanded, and/or enriched in the output composition produced.
In certain embodiments, the anti-idiotype antibody expands or enriches CAR-expressing cells infused into the composition. In some embodiments, the input composition comprises eukaryotic cells, such as mammalian cells. In some embodiments, the input composition contains human cells. In some embodiments, the infusion composition contains cells derived from blood, bone marrow, lymph, or lymphoid organs. In a specific embodiment, the input composition contains cells of the immune system, i.e. cells of the innate or adaptive immunity, such as myeloid cells or lymphoid cells, including lymphocytes, typically T cells and/or NK cells. In some embodiments, the input composition contains stem cells, such as pluripotent and multipotent stem cells, including induced pluripotent stem cells (ipscs). In particular embodiments, the input composition contains CD3 + And (3) cells. In certain embodiments, the input composition contains CD4 + And (3) cells. In some embodiments, the input composition contains CD8 + And (3) cells. In some embodiments, the input composition is a composition of cd4+ cells. In a specific embodiment, the input composition is a composition of cd8+ cells.
In some embodiments, the methods and agents are capable of stimulating T cells that lack or have down-regulated one or more natural signaling molecules, such as one or more co-stimulatory receptors or antigen receptors or cytokine receptors, but express a chimeric receptor (e.g., CAR) that is recognized by the anti-Id antibody. In some embodiments, the cells infused into the composition are low or negative with respect to surface expression of CD28 or other co-stimulatory molecules or other signaling molecules. Thus, in some embodiments, the provided agents and methods have certain advantages over certain other activating or stimulating agents or methods that may require or rely on surface expression of CD28 or other endogenous signal transduction molecules, which provide a desired signal and/or the degree of completion of such a signal, e.g., providing a co-stimulatory signal and/or achieving complete activation/in some embodiments, the provided agents and methods are advantageous in this regard as compared to anti-CD 3/anti-CD 28 agents (e.g., beads); in some aspects, the provided anti-ID antibodies have the advantage of being able to stimulate or achieve a desired effect, such as activation or proliferation of cells where CD28 or other natural signaling molecules are low or negative. In some aspects, only a single agent is used, both primary and secondary (co-stimulatory) signals are generated via the CAR by signal transduction of the CAR by stimulation with an anti-ID antibody. In some embodiments, the input composition comprises cd3+ cells that express low levels of CD28 or other endogenous signal transduction molecules. In some embodiments, the input composition comprises cd3+ cells that are CD28 negative or negative for other endogenous signal transduction molecules. In some embodiments, the anti-ID antibody stimulates activation and/or expansion of cells expressing low levels of CD28 or CD28 negative cells. In some embodiments, the cells are contacted with an anti-idiotype antibody or antigen-binding fragment immobilized or bound to a solid support. In some embodiments, the solid support is a bead. In some embodiments, the solid support is a surface of a well or plate (e.g., a cell culture plate). In some examples, the anti-ID antibody is soluble. In certain embodiments, the cells are not contacted with the anti-CD 3/anti-CD 28 conjugated agent prior to contacting the cells with the anti-idiotype antibody or antigen-binding fragment.
In certain embodiments, the anti-idiotype antibody is applied to, contacted with, or incubated with an input composition of a cell that has been transduced or transfected with a nucleotide encoding a CAR. In particular embodiments, incubating, treating, and/or contacting the input cells with an anti-idiotype antibody results in expansion and/or enrichment of the CAR-expressing cells. In particular embodiments, incubating, treating, and/or contacting the input cells with the anti-idiotype antibody does not result in the expansion and/or enrichment of cells that do not express the CAR. In particular embodiments, incubating, treating, and/or contacting the input cells with an anti-idiotype antibody results in at least 50%, at least 75%, at least 85%, at least 90%, at least 95%, at least 99%, at least 99.9%, or at least 99.99% less expansion and/or enrichment of cells that do not express the CAR than expansion and/or enrichment of cells that express the CAR. In some embodiments, the anti-idiotype antibodies provided herein are used to amplify CAR-expressing cells of an input composition that has undergone low transduction efficiency and/or low transfection efficiency and/or contains low CAR-expressing cells. In certain embodiments, the anti-idiotype antibody selectively expands and/or enriches cells expressing the CAR.
Some embodiments contemplate that the anti-idiotype antibody is more effective for amplifying and/or enriching cells having a low transduction efficiency or a low transfection efficiency of an input composition and/or cells having a low amount of CAR expression than by amplifying and/or enriching cells by means of polyclonal stimulation (e.g., anti-CD 3 and/or anti-CD 28 antibody stimulation). In particular embodiments, polyclonal stimulation results in expansion of CAR-expressing cells and non-CAR-expressing cells in the input composition, and thus, in some embodiments, CAR-expressing cells may not be enriched, particularly when the input composition has a low number of CAR-expressing cells. Conversely, in some embodiments, incubation with the anti-idiotype antibody results in selective expansion of the CAR-expressing cells, and thus, in certain embodiments, will result in selective expansion and/or enrichment of the CAR-expressing cells. In some embodiments, incubating, contacting, and/or treating the input cells with an anti-idiotype antibody results in more enrichment and/or expansion of CAR-expressing cells than by polyclonal stimulation.
In particular embodiments, the anti-idiotype antibody is usedGrowing, applying and/or contacting an input cell that is transfected and/or transduced with a lower amount of viral particles than an input cell expanded and/or enriched by polyclonal stimulation, which has a lower ratio of copies of viral vector particles to cells and/or a lower Infection Unit (IU). For example, in some embodiments, the input composition incubated with the anti-idiotype antibody is produced with 0.5, 1, 2, 3, 4, 5, 10, 15, 20, 30, 40, 50, or 60 IU/cell of the viral vector particle transduced cells less (or at least less) than the input composition amplified and/or enriched by polyclonal stimulation. In some embodiments, the input composition incubated with the anti-idiotype antibody is incubated with less (or at least less) 1x 10 than the input composition amplified and/or enriched by polyclonal stimulation 5 IU/mL、5x 10 5 IU/mL、1x 10 6 IU/mL、5x 10 6 IU/mL、6x 10 6 IU/mL、7x 10 6 IU/mL、8x 10 6 IU/mL、9x 10 6 IU/mL or 1x 10 7 Viral vector particle transduced cells at IU/mL titer.
In particular embodiments, transduced cells with high IU/cells will result in high transduction efficiency, but in some embodiments, transfected cells with high Vector Copy Number (VCN) may also result, which may present a safety risk and may not meet regulatory standards. In particular embodiments, decreasing transduced IU/cells will decrease transduction efficiency, but will decrease VCN. In particular embodiments, increasing the transduced IU/cells of the cell will increase transduction efficiency, but will also increase VCN.
In some embodiments, the input composition contains a population of cells that have been transduced or transfected with one or more nucleic acids encoding a CAR that has been bound or recognized by an anti-idiotype antibody, or cells derived from cells that have been transduced or transfected. In some embodiments, the input composition contains less than 80%, less than 75%, less than 70%, less than 65%, less than 60%, less than 55%, less than 50%, less than 45%, less than 40%, less than 35%, less than 30%, less than 25%, less than 20%, less than 15%, less than 10%, less than 5%, or less than 1% of CAR-expressing cells. In particular embodiments, cells from the input composition have been transfected or transduced as described in section III. In certain embodiments, the input cells contain a population of cells that have been transduced or transfected with one or more nucleic acids encoding an anti-CD 19CAR (such as an anti-CD 19CAR containing scFv that is derived and/or derived from an anti-CD 19 antibody (such as antibody SJ25C1 or FMC 63), or cells derived from cells that have been transduced or transfected.
In particular embodiments, the incubation, contacting or treatment of cells infused with the composition with an anti-idiotype antibody is performed under conditions for stimulating, expanding and/or activating the cells, which may include one or more of the following: specific media, temperature, oxygen content, carbon dioxide content, time, agents, e.g., nutrients, amino acids, antibiotics, ions, and/or stimulatory factors, such as cytokines, chemokines, antigens, binding partners, fusion proteins, recombinant soluble receptors, and any other agents intended to activate cells.
In some embodiments, the cells infused with the composition have been transfected or transduced with a nucleic acid comprising a gene encoding a CAR, and the cells are contacted, incubated, or treated with an anti-idiotype antibody that binds or recognizes the recombinant receptor. In some embodiments, after the cells infused with the composition transduce or transfect nucleic acid encoding the CAR, the cells are treated, incubated, or contacted with an anti-idiotype antibody. In particular embodiments, cells that are infused with the composition are treated, incubated, or contacted with the anti-idiotype antibody immediately after the cells that are infused with the composition have been transduced or transfected, within about 1 minute, within about 5 minutes, within about 30 minutes, within about 1 hour, within about 2 hours, within about 4 hours, within about 6 hours, within about 8 hours, within about 12 hours, within about 24 hours, within about 2 days, within about 3 days, within about 4 days, within about 5 days, within about 6 days, within about 1 week, within about 2 weeks, within about 3 weeks, within about 4 weeks, within about 5 weeks, or within about 6 weeks.
In some embodiments, cells infused into the composition are treated, incubated, and/or contacted with soluble anti-idiotype antibodies, with uncrosslinked antibodies, and/or with antibodies that are not bound or attached to a solid support.
In some embodiments, the methods result in proliferation, activation, stimulation, cytokine release, or other functional outcome, such as upregulation of an activation marker or cytokine release or production, of cells expressing a chimeric receptor (such as a CAR recognized by an anti-idiotype antibody). In some aspects, the extent to which such proliferation or other functional response or readout is induced in such cells is similar to or greater than that induced by incubating the cells with agents and/or conditions that stimulate T cell proliferation (e.g., anti-CD 3/CD28 beads and/or cross-linked anti-CD 3). In some aspects, the methods do not involve cross-linking of anti-idiotype antibodies. In some aspects of any embodiment, the anti-idiotype agent is capable of inducing a particular proliferation or functional outcome or degree thereof without cross-linking the anti-idiotype antibody. In some aspects, the anti-idiotype agents herein are advantageous in terms of their ability to stimulate or elicit specific functional consequences for T cells or other immune cells expressing the target receptor without the need to crosslink the anti-Id antibody or use a second agent. In some aspects, the results are achieved with anti-idiotype antibodies in soluble or plate-bound form. In some aspects, the results are achieved with anti-idiotype antibodies conjugated to beads.
In particular embodiments, cells that are to be infused with the composition are treated, incubated, and/or contacted with 10pg/ml to 100 μg/ml, 1pg/ml to 1ng/ml, 1ng/ml to 1 μg/l, 100ng/ml to 1.0 μg/ml, 1ng/ml to 100ng/ml, 10ng/ml to 1.0 μg/ml, 100ng/ml to 10pg/ml, 250ng/ml to 10 μg/ml, 250pg/ml to 1ng/ml, 1 μg/ml to 10 μg/ml, 250ng to about 2.5 μg/ml, or 1 μg/ml to 10 μg/ml.
In some embodiments, the anti-idiotype antibody or antigen-binding fragment thereof is immobilized to a solid support, optionally comprising or conjugated to an agent comprising a plurality of binding sites capable of reversibly binding to the anti-idiotype antibody or antigen-binding fragment thereof. In some embodiments, the solid support is a surface of a plate or well. In some embodiments, the anti-idiotype antibody or antigen-binding fragment thereof is immobilized to a soluble agent, optionally or comprising a plurality of binding sites capable of reversibly binding to the anti-idiotype antibody or antigen-binding fragment thereof. In some embodiments, the agent comprises a streptavidin mutein. In one exemplary embodiment, the anti-idiotype antibody comprises a streptavidin binding peptide or other streptavidin binding moiety that is capable of binding to a streptavidin or streptavidin mutein molecule present or immobilized on a soluble reagent, in some cases, that can be dissociated in the presence of a competing substance such as biotin. Examples of such systems include those described in PCT published patent application No. WO 2015/158868.
In particular embodiments, cells that are input into the composition are treated, incubated, and/or contacted with an anti-idiotype antibody that is attached, bound, coated, and/or conjugated to a solid surface or support (e.g., a plate or well). In certain embodiments, the anti-idiotype antibody is attached, bound, coated and/or conjugated to a solid surface or support by incubating the solid surface or support with a concentration of the anti-idiotype antibody. In specific embodiments, the solid surface or support is incubated with 10ng/ml to 100 μg/ml, 100ng/ml to 1.0 μg/ml, 250ng/ml to 10 μg/ml, 250ng/ml to 1 μg/ml, 1 μg/ml to 10 μg/ml, 250ng/ml to 2.5 μg/ml, or 1 μg/ml to 10 μg/ml of the anti-idiotype antibody. In some embodiments, the solid surface or support is incubated with 250ng/ml to 10 μg/ml of anti-idiotype antibody. In certain embodiments, the solid surface or support is incubated with (or with about) 0.25 μg/ml, 0.5 μg/ml, 1.0 μg/ml, 1.25 μg/ml, 2 μg/ml, 2.5 μg/ml, 5 μg/ml, or 10 μg/ml of anti-idiotype antibody.
In some embodiments, the incubation is at least or about at least 5 minutes, 10 minutes, 30 minutes, 60 minutes, 2 hours, 6 hours, 12 hours, 24 hours, 36 hours, 48 hours, 72 hours, or 96 hours. In some embodiments, the input composition comprises less than or less than about 60%, less than or less than about 50%, less than or less than about 40%, less than or less than about 30%, less than or less than about 20%, or less than about 10% of CAR-expressing cells as a percentage of the total cells in the composition. In some embodiments, the number of CAR-expressing cells in the output composition is increased by greater than 1.2-fold, 1.5-fold, 2.0-fold, 3.0-fold, 4.0-fold, 5.0-fold, 10-fold, or more as compared to the number of CAR-expressing cells in the input composition; and/or the percentage of CAR-expressing cells in the output composition is increased by greater than 10%, 20%, 40%, 50%, 60%, 70%, 80% or more as compared to the total cells in the composition. In some embodiments, the CAR-expressing cells are not selected or enriched for cells prior to incubation. In some embodiments, the target antibody or antigen-binding fragment thereof comprises the heavy chain variable region set forth in SEQ ID NO. 23 and/or the light chain variable region set forth in SEQ ID NO. 24. In some embodiments, the target antibody or antigen-binding fragment thereof comprises the heavy chain variable region set forth in SEQ ID NO. 30 and/or the light chain variable region set forth in SEQ ID NO. 31.
In certain embodiments, cells from the input composition (e.g., comprising CAR-expressing cells) are contacted or incubated with the anti-idiotype antibody for an amount of time to expand one or more cells of the input composition, such as to expand cells of the input composition that express the recombinant receptor. In particular embodiments, cells from the input composition are contacted, incubated, or treated with the anti-idiotype antibody for at least about 12 hours, at least about 24 hours, at least about 2 days, at least about 3 days, at least about 4 days, at least about 5 days, at least about 6 days, at least about 7 days, at least about 8 days, at least about 9 days, at least about 10 days, at least about 11 days, at least about 12 days, at least about 13 days, at least about 14 days, at least about 3 weeks, or at least about 4 weeks. In particular embodiments, cells from the input composition are contacted, incubated, or treated with the anti-idiotype antibody for less than about 1 day, less than about 2 days, less than about 3 days, less than about 4 days, less than about 5 days, less than about 6 days, or less than or about 12 days. In some embodiments, cells from the input composition are contacted, incubated, or treated with the anti-idiotype antibody for about 1 day to about 14 days, about 3 days to 7 days, or 4 days to 6 days.
In particular embodiments, cells from the input composition, e.g., comprising CAR-expressing cells, are incubated, contacted, or treated with the anti-idiotype antibody at a temperature above room temperature to expand cells of the input composition that express the recombinant receptor. In some embodiments, the treating, incubating, or contacting is performed at a temperature greater than about 25 ℃, such as typically greater than or greater than about 32 ℃, 35 ℃, or 37 ℃. In some embodiments, the treating, contacting, or incubating is performed at or at a temperature of about 37 ℃ ± 2 ℃, such as at or at a temperature of about 37 ℃.
In some embodiments, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, at least 99.9%, about 100%, or 100% of the cells of the output composition express the CAR.
In particular embodiments, the number of cells expressing the CAR in the output composition that are incubated, treated, and/or contacted with the anti-idiotype antibody is at least 1%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 95%, at least 100%, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, at least 15-fold, at least 20-fold, at least 25-fold, at least 50-fold, or at least 100-fold greater than the number of cells expressing the CAR in the input composition.
In particular embodiments, the percentage of cells expressing the CAR in the output composition that are incubated, treated, and/or contacted with the anti-idiotype antibody is at least 1%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 95%, at least 100%, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, at least 15-fold, at least 20-fold, at least 25-fold, at least 50-fold, or at least 100-fold greater than the number of cells expressing the CAR in the input composition.
In some embodiments, the number of cells expressing the CAR in the output composition that is incubated, treated, and/or contacted with the anti-idiotype antibody is at least 1%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 95%, at least 100%, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, at least 15-fold, at least 20-fold, at least 25-fold, at least 50-fold, or at least 100-fold greater than the number of cells in the output composition that is subjected to polyclonal stimulation (incubated with the anti-CD 3 and anti-CD 28 antibody).
In certain embodiments, the percentage of cells expressing the CAR in the output composition that are incubated, treated, and/or contacted with the anti-idiotype antibody is at least 1%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 95%, at least 100%, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, at least 15-fold, at least 20-fold, at least 25-fold, at least 50-fold, or at least 100-fold greater than the number of cells in the output composition that are subjected to polyclonal stimulation (incubated with the anti-CD 3 and anti-CD 28 antibody).
In some embodiments, the CAR-expressing cells in the output composition that are incubated, treated, and/or contacted with the anti-idiotype antibody contain at least 1%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 95%, or at least 99% less VCN than cells of the output composition that are subjected to polyclonal stimulation (e.g., incubation with anti-CD 3 and anti-CD 28 antibodies). In some embodiments, the average VCN of the CAR-expressing cells in the output composition does not exceed or exceed about 10, 5, 4, 2.5, 1.5, or 1.
In some embodiments, such methods can be used as part of manufacturing, analysis, and/or quality control methods, for example, associated with the production of cell therapies (such as CAR T cells) that express recombinant polypeptides containing antibodies or fragments thereof that are recognized by anti-idiotype antibodies, for testing purposes, including testing the expression and/or efficacy of engineered receptors in cells, for example, engineered for therapy in an individual. In certain embodiments, the cell composition can be tested at any stage in the process of generating T cells that express the CAR. In particular embodiments, cell samples may be collected from the cell composition at any stage of the process and stored, for example, by cryopreservation and/or cryopreservation, for later testing and/or analysis. The compositions tested may be pharmaceutical compositions, for example, including those comprising cells and pharmaceutically acceptable excipients (directives) and/or cryopreservatives.
In some embodiments, the anti-idiotype antibody stimulates cells expressing a target antibody (e.g., CAR) in vivo. Particular embodiments contemplate that CAR-T cell therapy is effective in the treatment of cancer and other diseases and disorders. However, in some cases, available methods of CAR-T cell therapy may not always be entirely satisfactory. For example, in some embodiments, the exposure and persistence of CAR-expressing cells in a subject decreases or declines over time. However, observations indicate that in some cases, increased exposure of CAR-expressing cells can improve the efficacy and therapeutic outcome of CAR-T cell therapy. Thus, in some embodiments, an anti-idiotype antibody is administered to enhance, boost, and/or increase the persistence and/or expansion of a CAR-expressing cell.
In certain embodiments, the anti-idiotype antibody is administered to a subject, such as a subject that has previously been administered a therapeutic cell composition comprising a CAR-expressing cell. In some embodiments, administration of the anti-idiotype antibody to the subject facilitates re-expansion of CAR-expressing cells in the subject, which in some cases can reach or exceed an initial peak expansion level prior to administration of the anti-idiotype antibody. In some embodiments, when the level of the CAR-expressing cells is reduced or undetectable, an anti-idiotype antibody is administered to modulate the expansion and/or persistence of the CAR-expressing cells. In some embodiments, for example, the CAR-expressing cells reamplified by the anti-idiotype antibody exhibit increased potency in the subject to whom it is administered as compared to the potency prior to administration of the anti-idiotype antibody.
In certain embodiments, administration of the anti-idiotype antibody increases or enhances the persistence of CAR-expressing cells in the subject. In some embodiments, the CAR-expressing cells are detectable in the subject 7 days, 14 days, 21 days, 28 days, 35 days, 42 days, 49 days, 56 days, 63 days, 2 months, 3 months, 4 months, 5 months, 6 months, or more than 6 months after administration of the anti-idiotype antibody. In some aspects, the increased exposure of the subject to the cells comprises increased expansion of the cells and/or expansion of the cells.
In some embodiments, the CAR-expressing cells are expanded in the subject after administration of the anti-idiotype antibody. In particular embodiments, administration of the anti-idiotype antibody results in a maximum concentration of at least 100, 500, 1000, 1500, 2000, 5000, 10,000, or 15,000 CAR-encoding nucleic acid copies per microgram of DNA, or at least 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, or 0.9 CAR-expressing cells per microliter in the blood or serum or other bodily fluid or organ or tissue of the subject. In some embodiments, the CAR-expressing cells are detected as at least 10%, 20%, 30%, 40%, 50% or 60% of total PBMCs in the subject's blood, and/or at such a level for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 24, 36, 48 weeks or 52 weeks after administration of the anti-idiotype antibody, or for 1, 2, 3, 4, 5 years or more after administration of the anti-idiotype antibody. In some aspects, administration of an anti-idiotype antibody results in at least a 2-fold, at least a 4-fold, at least a 10-fold, or at least a 20-fold increase in copies of nucleic acid encoding a recombinant receptor (e.g., CAR) per microgram of DNA in, for example, serum, plasma, blood, or tissue (e.g., a tumor sample) of a subject. In specific embodiments, administration of the anti-idiotype antibody results in an increase in the number of CAR-expressing cells circulating in the subject of at least 2-fold, at least 4-fold, at least 10-fold, or at least 20-fold.
In some aspects, at least about 1x 10 after administration of the anti-idiotype antibody 2 At least about 1x 10 3 At least about 1x 10 4 At least about 1x 10 5 Or at least about 1x 10 6 Or at least about 5x 10 6 Or at least about 1x 10 7 Or at least about 5x 10 7 Or at least about 1x 10 8 At least 10, 25, 50, 100, 200, 300, 400 or 500 or 1000 (e.g., at least 10 per microliter) CAR-expressing cells per microliter are detectable or present in the subject or a liquid, plasma, serum, tissue or compartment thereof, such as in the blood, e.g., in peripheral blood or a disease site. In some embodiments, such an amount or concentration of cells is detectable in the subject at least about 20 days, at least about 40 days, or at least about 60 days, or at least about 3, 4, 5, 6, 7, 8, 9, 10, 11 months, or 12 months, or at least 2 years, or 3 years after administration of the anti-idiotype antibody.
Various delivery systems are known and can be used to administer anti-idiotype antibodies. In certain embodiments, the anti-idiotype antibody is administered by encapsulation in and/or attachment to liposomes, microparticles, and microcapsules. Methods of administering anti-idiotype antibodies include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes. The anti-idiotype antibody may be administered by any convenient route, e.g., infusion, bolus injection, absorption via the epithelium or mucosa (e.g., oral, rectal, and intestinal mucosa, etc.), and may be administered with other bioactive agents. Administration may be systemic or local. Pulmonary administration may also be employed, for example, through the use of an inhaler or nebulizer, and formulation with a nebulizer. In certain embodiments, the anti-idiotype antibody is delivered in a vesicle, particularly a liposome (Langer, 1990,Science 249:1527-1533), such as a cationic liposome (WO 98140052).
In some embodiments, methods of producing a cellular composition are provided, the methods comprising introducing a nucleic acid molecule encoding a CAR into a cell, thereby generating an input composition, and incubating the input composition with an anti-idiotype antibody or antigen-binding fragment thereof specific for an antigen-binding domain of the CAR, thereby producing the cellular composition. In some embodiments, the CAR comprises a target antibody or antigen-binding fragment thereof that specifically binds to CD 19. In some embodiments, the target antibody is antibody SJ25C1 or FMC63 or an antigen binding fragment thereof. In some embodiments, the anti-idiotype antibody or antigen-binding fragment thereof is an anti-idiotype antibody or antigen-binding fragment thereof described herein. In some embodiments, the anti-idiotype antibody or antigen-binding fragment thereof is an agonist of the CAR. In some embodiments, introducing comprises introducing the nucleic acid molecule into the cell by viral transduction, transposition, electroporation, or chemical transfection. In some embodiments, introducing comprises introducing the nucleic acid molecule into the cell by transduction with a retroviral vector comprising the nucleic acid molecule, by transduction with a lentiviral vector comprising the nucleic acid molecule, by transposition with a transposon comprising the nucleic acid molecule, or by electroporation or transfection of a vector comprising the nucleic acid molecule.
In some embodiments, the method further comprises the step of stimulating or activating the cell prior to introducing the nucleic acid molecule encoding the CAR. In some embodiments, activating the cells comprises contacting the cells with a CD3 agonist and optionally a CD28 agonist. In some embodiments, activating the cells comprises contacting the cells with an agent comprising an agonistic anti-CD 3 antibody and an anti-CD 28 antibody. In some such embodiments, the method comprises incubating or contacting the cell with an anti-idiotype antibody or antigen-binding fragment thereof during at least a portion of the contacting with anti-CD 3/anti-CD 28 and/or during at least a portion of the introducing nucleic acid encoding the CAR. In some embodiments, the incubation is performed under conditions in which the anti-idiotype antibody or antigen-binding fragment thereof binds to the CAR, thereby inducing or modulating a signal in one or more cells of the input composition. In some embodiments, the cells comprise T cells.
In some such embodiments, the T cells comprise cd4+ and/or cd8+ T cells. In some embodiments, the anti-idiotype antibody or antigen-binding fragment thereof is immobilized to a solid support, optionally comprising or conjugated to an agent comprising a plurality of binding sites capable of reversibly binding to the anti-idiotype antibody or antigen-binding fragment thereof. In some embodiments, the anti-idiotype antibody or antigen-binding fragment thereof is immobilized to a soluble agent, optionally or comprising a plurality of binding sites capable of reversibly binding to the anti-idiotype antibody or antigen-binding fragment thereof. In some embodiments, the agent comprises a streptavidin mutein. In one exemplary embodiment, the anti-idiotype antibody comprises a streptavidin binding peptide or other streptavidin binding moiety that is capable of binding to a streptavidin or streptavidin mutein molecule present or immobilized on a soluble reagent, in some cases, that can be dissociated in the presence of a competing substance such as biotin. Examples of such systems include those described in PCT published patent application No. WO 2015/158868. In some embodiments, the incubation is at least or about at least 5 minutes, 10 minutes, 30 minutes, 60 minutes, 2 hours, 6 hours, 12 hours, 24 hours, 36 hours, 48 hours, 72 hours, or 96 hours. In some embodiments, the input composition comprises less than or less than about 60%, less than or less than about 50%, less than or less than about 40%, less than or less than about 30%, less than or less than about 20%, or less than about 10% of CAR-expressing cells as a percentage of the total cells in the composition. In some embodiments, the number of CAR-expressing cells in the output composition is increased by greater than 1.2-fold, 1.5-fold, 2.0-fold, 3.0-fold, 4.0-fold, 5.0-fold, 10-fold, or more as compared to the number of CAR-expressing cells in the input composition; and/or the percentage of CAR-expressing cells in the output composition is increased by greater than 10%, 20%, 40%, 50%, 60%, 70%, 80% or more as compared to the total cells in the composition. In some embodiments, the CAR-expressing cells are not selected or enriched for cells prior to incubation. In some embodiments, the target antibody or antigen-binding fragment thereof comprises the heavy chain variable region set forth in SEQ ID NO. 23 and/or the light chain variable region set forth in SEQ ID NO. 24. In some embodiments, the target antibody or antigen-binding fragment thereof comprises the heavy chain variable region set forth in SEQ ID NO. 30 and/or the light chain variable region set forth in SEQ ID NO. 31.
In some embodiments, a method of monitoring the activity of a CAR comprising a target antibody, such as antibody SJ25C1 or FMC63, or an antigen binding fragment thereof, is provided, the method comprising the steps of: incubating a sample comprising T cells transduced with a CAR with an agonistic anti-idiotype antibody or antigen binding fragment thereof that targets or binds the CAR; and/or determining the presence, absence or amount of activation, stimulation and/or expansion of CAR T cells, thereby monitoring the activity of the CAR-T cells. In some embodiments, such methods can be used to verify a CAR, in which case the method can include c) verifying a CAR based on the level of activation, stimulation, and/or expansion of the CAR-T cells.
In some embodiments, activation, stimulation, and/or expansion of CAR T cells is assessed by determining the viability, proliferation, and/or expression of T cell activation markers in CAR T cells after incubation with an anti-idiotype antibody for a period of time. In some embodiments, the viability of the CAR T cells is assessed by calculating the percentage of live T cells transduced with the CAR to total T cells after incubation with the anti-idiotype antibody. In some embodiments, proliferation of CAR T cells is assessed by dye dilution, which is used to stain CAR T cells prior to incubation with anti-idiotype antibodies. In some embodiments, expression of the T cell activation marker is assessed by flow cytometry and staining for antibodies that recognize the T cell activation marker. In some embodiments, the T cell activation marker is selected from the group consisting of: CD25, CD26, CD27, CD28, CD30, CD69, CD71, CD134, CD137 and CD154. In some embodiments, the incubation time is from about 1 day to about 10 days (e.g., any of about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 days, including any range between these values)). In some embodiments, the target antibody or antigen-binding fragment thereof comprises the heavy chain variable region set forth in SEQ ID NO. 23 and/or the light chain variable region set forth in SEQ ID NO. 24. In some embodiments, the target antibody or antigen-binding fragment thereof comprises the heavy chain variable region set forth in SEQ ID NO. 30 and/or the light chain variable region set forth in SEQ ID NO. 31.
In some embodiments, a method of monitoring a formulation of a CAR T cell is provided, wherein the CAR comprises a target antibody, such as antibody SJ25C1 or FMC63, or an antigen binding fragment thereof, the method comprising a) incubating a portion of the formulation with an agonistic anti-idiotype antibody or antigen binding fragment thereof that targets or binds the CAR; and b) determining the presence, absence or amount of activation, stimulation and/or expansion of CAR T cells. In some embodiments, the CAR-T cell formulation can be a cell that is produced or manufactured under the particular conditions desired to be tested. In some embodiments, monitoring is performed in conjunction with a release assay (release assay), such as for validating the cells prior to administration to a subject. In some aspects, the method further comprises c) validating the formulation based on the level of activation of CAR T cells. In some embodiments, activation of CAR T cells in the formulation is assessed by determining the viability, proliferation, and/or expression of T cell activation markers in CAR T cells after incubation with an anti-idiotype antibody for a period of time. In some embodiments, the viability of the CAR T cells is assessed by calculating the percentage of live T cells transduced with the CAR to total T cells after incubation with the anti-idiotype antibody. In some embodiments, proliferation of CAR T cells is assessed by dye dilution, which is used to stain CAR T cells prior to incubation with anti-idiotype antibodies. In some embodiments, expression of the T cell activation marker is assessed by flow cytometry and staining for antibodies that recognize the T cell activation marker. In some embodiments, the T cell activation marker is selected from the group consisting of: CD25, CD26, CD27, CD28, CD30, CD69, CD71, CD134, CD137 and CD154. In some embodiments, the incubation time is from about 1 day to about 10 days (e.g., any of about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 days, including any range between these values)). In some embodiments, the target antibody or antigen-binding fragment thereof comprises the heavy chain variable region set forth in SEQ ID NO. 23 and/or the light chain variable region set forth in SEQ ID NO. 24. In some embodiments, the target antibody or antigen-binding fragment thereof comprises the heavy chain variable region set forth in SEQ ID NO. 30 and/or the light chain variable region set forth in SEQ ID NO. 31.
C. Use in cell inactivation/depletion
In some embodiments, the provided anti-idiotype antibodies or antigen-binding fragments thereof are antagonists and/or exhibit specific activity to inhibit, ablate, and/or deplete (e.g., by antibody-dependent cell-mediated cytotoxicity (ADCC) killing) cells expressing a target antibody, such as an anti-CD 19 antibody (e.g., antibody SJ25C1 or FMC 63) or antigen-binding fragment thereof. Also provided are methods involving the use of the provided anti-idiotype antibodies and molecules (such as conjugates and complexes) containing one or more such anti-idiotype antibodies for the inactivation, ablation and/or depletion of CAR T cells, wherein the CAR comprises a target antibody, such as an anti-CD 19 antibody (e.g., antibody SJ25C1 or FMC 63) or an antigen binding fragment thereof.
In some embodiments, the methods comprise treating, contacting, and/or incubating a composition and/or sample comprising T cells transduced with a CAR with an anti-idiotype antibody. In certain embodiments, the method further comprises detecting whether the CAR T cell is inactivated, such as by assessing the viability, proliferation, and/or expression of an activation marker in the CAR T cell. In some embodiments, the method is associated with a therapy comprising administering CAR T cells. In some embodiments, the method comprises administering to the individual an anti-idiotype antibody. In one embodiment, the anti-idiotype antibody or conjugate is used to ablate and/or deplete (e.g., kill) CAR T cells in an individual. In some embodiments, the target antibody is an anti-CD 19 antibody. In some embodiments, the target antibody is or is derived from antibody SJ25C1 or FMC63 or antigen binding fragment thereof. In some embodiments, the target antibody or antigen-binding fragment thereof comprises the heavy chain variable region set forth in SEQ ID NO. 23 and/or the light chain variable region set forth in SEQ ID NO. 24. In some embodiments, the target antibody or antigen-binding fragment thereof comprises the heavy chain variable region set forth in SEQ ID NO. 30 and/or the light chain variable region set forth in SEQ ID NO. 31.
In some embodiments, an anti-idiotype antibody is administered to deplete, reduce, and/or decrease the number of CAR-expressing cells in the subject. In particular embodiments, administration of the anti-idiotype antibody depletes, reduces, and/or reduces the amount of CAR-expressing cells (e.g., circulating CAR-T cells) by at least 25%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 99%, at least 99.9%, 100%, or about 100%. In certain embodiments, the depletion, reduction, and/or decrease is related to the amount of CAR-expressing cells in the subject prior to administration of the anti-idiotype antibody. In particular embodiments, the depletion, reduction, and/or decrease is associated with the amount of CAR-expressing cells in the subject to whom the anti-idiotype antibody is not administered. In some embodiments, no CAR-expressing cells are detected in the subject after administration of the anti-idiotype antibody. In specific embodiments, the anti-idiotype antibody is a human antibody or a humanized antibody.
In some embodiments, methods of inactivating CAR T cells are provided, wherein the CAR comprises a target antibody, such as antibody SJ25C1 or FMC63, or an antigen binding fragment thereof, comprising incubating a sample comprising CAR T cells with an antagonistic anti-idiotype antibody or antigen binding fragment thereof that targets the CAR, thereby inactivating CAR T cells in the sample. In some embodiments, the anti-idiotype antibody is used in an amount sufficient to attenuate CAR T cell activation in the sample. In some embodiments, the anti-idiotype antibody is used in an amount sufficient to substantially inactivate CAR T cells in the sample. In some embodiments, the incubation with the anti-idiotype antibody results in the ablation and/or depletion of CAR T cells in the sample. In some embodiments, the anti-idiotype antibody is used in an amount sufficient to cause CAR T cell clearance in the sample. In some embodiments, the target antibody or antigen-binding fragment thereof comprises the heavy chain variable region set forth in SEQ ID NO. 23 and/or the light chain variable region set forth in SEQ ID NO. 24. In some embodiments, the target antibody or antigen-binding fragment thereof comprises the heavy chain variable region set forth in SEQ ID NO. 30 and/or the light chain variable region set forth in SEQ ID NO. 31.
In some embodiments, an anti-idiotype antibody is administered to deplete, reduce, and/or decrease the activity of a CAR and/or CAR-expressing cells in a subject. In particular embodiments, administration of the anti-idiotype antibody reduces and/or reduces stimulation and/or activation of at least 25%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 99%, at least 99.9%, 100% or about 100% of the CAR and/or CAR-expressing cells. In certain embodiments, the reduction and/or decrease is associated with stimulation and/or activity of the CAR and/or CAR-expressing cells in the subject prior to administration of the anti-idiotype antibody. In particular embodiments, the reduction and/or decrease is associated with stimulation and/or activity of the CAR and/or CAR-expressing cells in a subject not administered the anti-idiotype antibody. In some embodiments, activity and/or stimulation refers to one or more aspects of CAR receptor or CAR T cell activity, and can be assessed by any suitable known means, including by any means provided herein. In some embodiments, no activity and/or stimulation of the CAR and/or CAR-expressing cells is detected in the subject following administration of the anti-idiotype antibody. In specific embodiments, the anti-idiotype antibody is a human antibody or a humanized antibody.
In some embodiments, the anti-idiotype antibody is administered to prevent, reduce, and/or reduce the binding and/or binding capacity of the CAR and/or the CAR-expressing cell to the antigen. In particular embodiments, administration of the anti-idiotype antibody reduces and/or reduces antigen binding of at least 25%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 99%, at least 99.9%, 100% or about 100% of the CAR and/or CAR-expressing cells. In certain embodiments, the reduction and/or decrease is associated with antigen binding and/or ability to bind to an antigen of the CAR and/or CAR-expressing cells in the subject prior to administration of the anti-idiotype antibody. In particular embodiments, the reduction and/or decrease is associated with antigen binding and/or the ability of the CAR and/or CAR-expressing cells to bind antigen in a subject not administered an anti-idiotype antibody. In specific embodiments, the anti-idiotype antibody is a human antibody or a humanized antibody.
In some embodiments, methods of ablating and/or depleting (e.g., killing) CAR T cells are provided, wherein the CAR comprises a target antibody, such as antibody SJ25C1 or FMC63, or an antigen binding fragment thereof, the method comprising incubating a sample comprising CAR T cells with an anti-idiotype antibody or antigen binding fragment thereof that targets the CAR, thereby ablating and/or depleting CAR T cells in the sample. In some embodiments, ablation and/or depletion is by means of antibody-dependent cell-mediated cytotoxicity (ADCC). In some embodiments, the anti-idiotype antibody is used in an amount sufficient to cause ablation and/or depletion of substantially all CAR T cells in the sample. In some embodiments, the target antibody or antigen-binding fragment thereof comprises the heavy chain variable region set forth in SEQ ID NO. 23 and/or the light chain variable region set forth in SEQ ID NO. 24. In some embodiments, the target antibody or antigen-binding fragment thereof comprises the heavy chain variable region set forth in SEQ ID NO. 30 and/or the light chain variable region set forth in SEQ ID NO. 31.
In some embodiments, a method of modulating CAR T cell therapy in an individual is provided, wherein the CAR comprises a target antibody, such as antibody SJ25C1 or FMC63, or an antigen binding fragment thereof, comprising administering to the individual an antagonistic anti-idiotype antibody or antigen binding fragment thereof that targets the CAR, thereby inactivating the CAR T cells. In some embodiments, the anti-idiotype antibody is administered in an amount sufficient to attenuate CAR T cell activation in the individual. In some embodiments, the anti-idiotype antibody is administered in an amount sufficient to substantially inactivate CAR T cells in the individual. In some embodiments, administration of the anti-idiotype antibody results in the ablation and/or depletion of CAR T cells in the individual. In some embodiments, the anti-idiotype antibody is administered in an amount sufficient to cause CAR T cell clearance in the individual. In some embodiments, the target antibody or antigen-binding fragment thereof comprises the heavy chain variable region set forth in SEQ ID NO. 23 and/or the light chain variable region set forth in SEQ ID NO. 24. In some embodiments, the target antibody or antigen-binding fragment thereof comprises the heavy chain variable region set forth in SEQ ID NO. 30 and/or the light chain variable region set forth in SEQ ID NO. 31.
In some embodiments, a method of modulating CAR T cell therapy in an individual is provided, wherein the CAR comprises a target antibody, such as antibody SJ25C1 or FMC63, or an antigen binding fragment thereof, the method comprising administering to the individual an anti-idiotype antibody immunoconjugate that targets the CAR, wherein the anti-idiotype antibody immunoconjugate comprises a cytotoxic agent. In some embodiments, the anti-idiotype antibody immunoconjugate is administered in an amount sufficient to attenuate CAR T cell therapy in the individual. In some embodiments, the anti-idiotype antibody immunoconjugate is administered in an amount sufficient to substantially stop CAR T cell therapy in the individual. In some embodiments, the anti-idiotype antibody immunoconjugate is administered in an amount sufficient to cause CAR T cell clearance in the individual. In some embodiments, the cytotoxic agent is selected from the group consisting of: chemotherapeutic agents or drugs, growth inhibitors, toxins (e.g., bacterial, fungal, plant or animal derived protein toxins, enzymatically active toxins, or fragments thereof), and radioisotopes. In some embodiments, the target antibody or antigen-binding fragment thereof comprises the heavy chain variable region set forth in SEQ ID NO. 23 and/or the light chain variable region set forth in SEQ ID NO. 24. In some embodiments, the target antibody or antigen-binding fragment thereof comprises the heavy chain variable region set forth in SEQ ID NO. 30 and/or the light chain variable region set forth in SEQ ID NO. 31.
Use in binding assays or methods
Provided herein are methods for assessing the presence or absence of a molecule in a sample that binds to a Chimeric Antigen Receptor (CAR), such as an extracellular domain of a CAR or a portion thereof containing an antigen binding domain. In some embodiments, the methods can be used to assess the presence or absence of a humoral response or antibody response in a subject to an administered cell therapy comprising a Chimeric Antigen Receptor (CAR). In some embodiments, the chimeric antigen receptor comprises a target antibody that is antibody FMC63 or an antigen binding fragment thereof. In some embodiments, the chimeric antigen receptor comprises a target antibody that is antibody SJ25C1 or an antigen binding fragment thereof. In some embodiments, an anti-idiotype antibody or antigen-binding fragment thereof specific for the extracellular domain of a CAR (such as any of those described herein) can be used as a positive control in the method.
In particular embodiments, the method comprises contacting the sample with an anti-idiotype antibody or antigen-binding fragment thereof specific for an extracellular domain of a CAR, wherein the concentration of the anti-idiotype antibody is 10ng/ml to 100 μg/ml, 100ng/ml to 1.0 μg/ml, 250ng/ml to 10 μg/ml, 250ng/ml to 1 μg/ml, 1 μg/ml to 10 μg/ml, 250ng to 2.5 μg/ml, or 1 μg/ml to 10 μg/ml. In some embodiments, the concentration of anti-idiotype antibody is 250ng/ml to 10 μg/ml. In certain embodiments, the concentration of anti-idiotype antibody is about 0.1 μg/ml, 0.25 μg/ml, 0.5 μg/ml, 1.0 μg/ml, 1.25 μg/ml, 2 μg/ml, 2.5 μg/ml, or 5 μg/ml of anti-idiotype antibody.
In some aspects, adoptive cell therapy may be associated with the development of an immune response in a subject to the administered cells and/or constructs. For example, in some cases, exposure to a chimeric receptor may be limited by a host immune response to recombinant receptors expressed by the administered cells, which may prematurely eliminate the administered cells. It was observed that even in certain subjects with B cell malignancies, typically immunocompromised, immune responses specific for receptor regions expressed by cells administered in adoptive cell therapy could be detected. For example, a subject (e.g., a human subject) administered cells genetically engineered with a CAR can generate a specific immune response against immunogenic regions of the chimeric region, including regions that can contain non-human sequences (e.g., murine scFv) and/or regions that contain a linkage between two domains or portions of the chimeric receptor (e.g., the transmembrane and costimulatory domains of the CAR).
In some embodiments, methods are provided that involve contacting or incubating a binding agent with a sample from a subject that has been administered a cell therapy comprising chimeric antigen receptor-engineered cells, wherein the binding agent is a protein comprising an extracellular domain of a CAR or a portion thereof (containing a target antibody or antigen-binding fragment thereof). In some embodiments, the method further comprises detecting whether a complex is formed between the binding agent and a molecule present in the sample, e.g., a binding molecule (such as an antibody), and/or detecting the presence or absence or level of such binding. In certain embodiments, the contacting or incubating is under conditions that allow the binding agent to bind to molecules present in the sample from the subject. In certain aspects, the methods can further be performed on a positive control sample containing an anti-idiotype antibody or antigen-binding fragment thereof specific for any CAR as described. In some embodiments, determining the presence, absence, or level of binding of the molecule to the binding agent may comprise: the binding or detection is compared to that of a positive control sample and a binding reagent.
In some embodiments, the cell therapy is or comprises a genetically engineered cell expressing an anti-CD 19CAR comprising a target antibody (which is antibody SJ25C1 or an antigen binding fragment thereof), wherein the binding agent comprises an extracellular domain of the CAR or a portion thereof (comprising an SJ25C1 antibody or an antigen binding fragment thereof). In some embodiments, the positive control comprises an anti-idiotype antibody as described in section i.a.
In some embodiments, the cell therapy is or comprises a genetically engineered cell expressing an anti-CD 19CAR comprising a target antibody (which is antibody FMC63 or an antigen binding fragment thereof), wherein the binding agent comprises the extracellular domain of the CAR or a portion thereof (comprising FMC63 antibody or an antigen binding fragment thereof). In some embodiments, the positive control comprises an anti-idiotype antibody as described in section I.B.
In some embodiments, the method comprises detecting whether a complex is formed between the binding agent and a molecule present in the sample, e.g., a binding molecule (such as an antibody), and/or detecting the presence or absence or level of such binding. In certain embodiments, the contacting or incubating is under conditions that allow the binding agent to bind to molecules present in the sample from the subject. In some aspects, the complex is detected by an immunoassay, optionally a sandwich assay or a bridging assay. For example, the immunoassay is an enzyme-linked immunosorbent assay (ELISA), chemiluminescence, electrochemiluminescence, a Surface Plasmon Resonance (SPR) based biosensor (e.g., BIAcore), flow cytometry, or Western blot. In some embodiments, the immunoassay is or includes mesoscale discovery.
In some aspects, the immunoassay is a sandwich assay or a bridging assay. In a sandwich or bridging assay, the binding reagent is a first binding reagent and detecting the presence or absence of a molecule or complex comprising the molecule comprises: contacting the complex formed by the first binding agent and the molecule with a second binding agent, wherein the second binding agent is an agent capable of binding the same or similar molecule as the first binding agent. In some embodiments, the second binding agent comprises an extracellular domain of a CAR or a portion thereof. In some aspects, the CAR extracellular domains, or portions thereof, of the first binding agent and the second binding agent are the same or substantially the same.
In some embodiments, the binding reagent (e.g., first and/or second binding reagent) is labeled with a detectable label or is capable of producing a detectable signal. The binding reagent (e.g., first and/or second binding reagent) is directly or indirectly linked to a detectable label. In some embodiments, the detectable label is or includes a fluorescent label, a chemiluminescent label, an electroluminescent label, a colorimetric label, a bioluminescent label, or a radioactive label. In some embodiments, the binding agent (e.g., first and/or second binding agent) is directly or indirectly linked to a sulphur tag. In some embodiments, at least one of the first and second binding reagents is labeled or capable of producing a detectable signal, and the other of the first and second binding reagents is attached or immobilized to a solid support. In some aspects, the first binding reagent is or is capable of being attached or immobilized to a solid support. Methods for attaching binding reagents directly or indirectly to solid supports are well known in the art. The attachment method typically involves non-specific adsorption of the binding reagent onto the solid support or covalent attachment of the binding reagent (typically via free amine groups) to chemically reactive groups (such as activated carboxyl, hydroxyl or aldehyde groups) on the solid support. The attachment method further comprises indirectly attaching the binding reagent to the solid support, for example by coating the solid support with a capture reagent (such as streptavidin), and adding an affinity-tagged binding reagent (such as a biotin-tagged reagent) to the solid support such that an interaction between the affinity tag (e.g., biotin) and the capture reagent (e.g., streptavidin) attaches the binding reagent to the solid support. In some embodiments, the first binding reagent is directly or indirectly linked to biotin. In some examples, the first soluble reagent is bound to a solid support coated with streptavidin. In some embodiments, the second binding reagent is directly or indirectly linked to a detectable label (optionally a sulphur tag).
In particular embodiments, the sample is contacted with a first binding reagent that is attached, bound, coated and/or conjugated to a solid surface or support (e.g., plate or well). In certain embodiments, the first binding reagent is attached, bound, coated and/or conjugated to a solid surface or support by indirectly attaching the binding reagent to the solid support, e.g., by coating the solid support with a capture reagent (such as streptavidin), and adding an affinity-labeled binding reagent (such as a biotin-labeled reagent) to the solid support such that the interaction between the affinity label (e.g., biotin) and the capture reagent (e.g., streptavidin) attaches the binding reagent to the solid support. In some embodiments, the sample is contacted with a second binding reagent that is directly or indirectly linked to the sulphur tag. In particular embodiments, the first and/or second binding reagent is used at a concentration of: anti-idiotype antibody of 10ng/ml to 100 μg/ml, 100ng/ml to 1.0 μg/ml, 250ng/ml to 10 μg/ml, 250ng/ml to 1 μg/ml, 1 μg/ml to 10 μg/ml, 250ng/ml to 2.5 μg/ml or 1 μg/ml to 10 μg/ml. In some embodiments, the solid surface or support is incubated with 250ng/ml to 10 μg/ml of anti-idiotype antibody. In certain embodiments, the solid surface or support is incubated with (or with about) 0.25 μg/ml, 0.5 μg/ml, 1.0 μg/ml, 1.25 μg/ml, 2 μg/ml, 2.5 μg/ml, 5 μg/ml, or 10 μg/ml of anti-idiotype antibody.
In some embodiments, the sample from a subject to whom a cell therapy comprising chimeric antigen receptor engineered cells has been administered is or comprises any body fluid sample from the subject. In some aspects, the sample is or comprises whole blood, serum, or plasma. In some embodiments, the sample is obtained from the subject within 1 hour to 1 year after (or about) the initiation of administration of the cell therapy or cell dose, such as within (or about) 6 hours, 12 hours, 24 hours, one week, two weeks, three weeks, one month, two months, three months, four months, five months, six months, seven months, eight months, nine months, ten months, eleven months, or twelve months. In some aspects, the sample is obtained from the subject after (or about) 1 month to 6 months after initiation of administration of the cell therapy, such as 2 months to 6 months or 2 months to 4 months after initiation of administration of the cell therapy, e.g., about or about 2, 3 months, 4 months, 5 months, or 6 months.
Article of manufacture
Also provided are articles of manufacture or kits comprising the provided anti-idiotype antibodies and/or compositions. In some embodiments, an article of manufacture comprising an anti-idiotype antibody or antigen-binding fragment thereof is provided. In some cases, the anti-idiotype antibody binds to an anti-CD 19 antibody or antigen-binding fragment thereof, or a chimeric antigen receptor comprising an anti-CD 19 antibody or antigen-binding fragment thereof. In some examples, the anti-CD 19 antibody is SJ25C1 or FMC63. In some aspects, conjugates comprising the anti-idiotype antibodies described herein are provided in a preparation or kit.
In some embodiments, the kit or article of manufacture comprises an anti-idiotype antibody or antigen-binding fragment thereof and a binding agent comprising an extracellular domain or portion of an extracellular domain of a Chimeric Antigen Receptor (CAR) to which the idiotype antibody binds (e.g., specifically binds). In some embodiments, the extracellular domain of the CAR is or comprises an anti-CD 19 antibody (e.g., FMC63 or SJ25C 1) or antigen binding fragment thereof.
In some embodiments, the binding reagent is a first binding reagent and the kit or article of manufacture further comprises a second binding reagent. In such examples, the second binding reagent is a reagent capable of binding the same or similar molecule as the first binding reagent. In some embodiments, the second binding agent comprises an extracellular domain of a CAR or a portion thereof. In some aspects, the CAR extracellular domains, or portions thereof, of the first binding agent and the second binding agent are the same or substantially the same.
In some embodiments, at least one of the binding reagent or the first and second binding reagents is attached to a label (e.g., a detectable label) such as the labels described herein. In some embodiments, at least one of the first and second binding reagents is attached to or capable of being attached to a solid support, such as the solid supports described herein. In some aspects, one of the first and second binding reagents is labeled or capable of producing a detectable signal, and the other of the first and second binding reagents is attached or immobilized to a solid support. In some embodiments, a binding reagent is provided as a kit or as part of a system described elsewhere herein for use in conjunction with an immunoassay (e.g., a sandwich or bridging assay). In some embodiments, the first binding reagent is bound to a solid support, optionally a streptavidin-coated solid support. In some embodiments, the second soluble protein is directly or indirectly linked to a detectable label (such as a sulphur tag).
In some embodiments, the kit further comprises an anti-idiotype antibody or antigen-binding fragment thereof. In some aspects, the anti-idiotype antibody binds to an anti-CD 19 antibody or antigen-binding fragment thereof, or a chimeric antigen receptor comprising an anti-CD 19 antibody or antigen-binding fragment thereof. In some examples, the anti-CD 19 antibody is SJ25C1 or FMC63. In some embodiments, an anti-idiotype antibody or antigen-binding fragment thereof is provided as a positive control sample. In some examples, the positive control sample forms a complex with first and second soluble proteins or reagents that contain a region of the extracellular domain of a Chimeric Antigen Receptor (CAR) that comprises a CD19 antibody or antigen binding fragment thereof.
In some embodiments, the kit or article of manufacture comprises reagents or components for performing any of the provided methods. In some embodiments, the article of manufacture or kit comprises one or more reagents or other substances, including secondary antibodies, affinity tags, capture reagents, buffers, diluents, signal detection agents, filters, needles, syringes, capillaries, and package insert with instructions for use, based on commercial, therapeutic, and user-friendly needs.
In some embodiments, the kit may be provided as an article of manufacture comprising packaging material for packaging the antibody or a composition thereof or one or more additional agents, such as binding agents or components. For example, the kit may include containers, bottles, tubes, vials, and any packaging material suitable for separating or organizing the kit components.
In some embodiments, the kit comprises one or more containers. Suitable containers include, for example, bottles, vials (e.g., dual chamber vials), syringes (such as single chamber or dual chamber syringes), and test tubes. One or more of the containers may be formed from various materials such as glass or plastic. The one or more containers hold a composition comprising an antibody or other reagent (e.g., a binding reagent) for use in the method. The articles or kits herein may comprise antibodies or reagents in separate containers or in the same container. In some embodiments, the one or more containers containing the composition may be disposable vials or multiple use vials, which in some cases may allow for the reuse of the reconstituted composition.
In some embodiments, the article of manufacture or kit may further comprise a second container comprising a suitable diluent. The article of manufacture or kit may further comprise other materials based on commercial, therapeutic, and user-friendly needs including other buffers, diluents, filters, needles, syringes, therapeutic agents, and/or package insert with instructions for use.
The article of manufacture may include a container and a label or package insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, IV solution bags, and the like. The container may be formed from a variety of materials such as glass or plastic. In some embodiments, the container contains a composition comprising an anti-idiotype antibody provided herein, alone or in combination with another composition, for effective treatment, prevention, and/or diagnosis of a disease or disorder. In some embodiments, the container has a sterile access port. Exemplary containers include intravenous solution bags, vials, including those having a stopper pierceable by an injection needle. The article of manufacture may comprise a first container having a composition therein, wherein the composition comprises an anti-idiotype antibody. Alternatively, or in addition, the article of manufacture may further comprise another or the same container comprising an acceptable buffer. It may further include other items such as other buffers, diluents, filters, needles and/or syringes.
In some embodiments, the article or kit comprises a solid support, including a solid support formed from glass (e.g., controlled pore glass), polysaccharide (e.g., agarose), polyacrylamide, polystyrene, polyvinyl alcohol, nitrocellulose, cellulose, nylon, silicone, and other materials well known in the art, for use in a solid support for direct or indirect attachment of a binding agent as described. Solid supports included in the articles or kits provided herein include, but are not limited to, beads, columns (e.g., chromatography columns, etc.), arrays (e.g., microarrays, nanoarrays, etc.), assay plates, cartridges, rods, filters, strips, or any other solid support described herein.
In some embodiments, the article of manufacture or kit may further comprise a second container comprising a suitable diluent. The article of manufacture or kit may further comprise other materials based on commercial, therapeutic, and user-friendly needs including other buffers, diluents, filters, needles, syringes, therapeutic agents, and/or package insert with instructions for use.
In some embodiments, the kit may optionally include instructions. The instructions generally include tangible expressions describing the antibodies and optionally other components included in the kit (e.g., binding reagents), as well as methods of using the antibodies and/or other components in (or in conjunction with) any of the uses or methods. In some embodiments, the instructions are provided as a label or package insert located on or associated with the container. In some embodiments, the instructions may indicate instructions for reconstitution and/or use of the composition.
In some embodiments, instructions for using an anti-idiotype antibody to detect a SJ25C1 antibody or antigen-binding fragment thereof, or a chimeric antigen receptor comprising a SJ25C1 antibody or antigen-binding fragment thereof, are provided, e.g., according to (or in conjunction with) any of the methods or assays. In some examples, instructions for using an anti-idiotype antibody to select or enrich for engineered cells from a population of cells that express a Chimeric Antigen Receptor (CAR) comprising antibody SJ25C1 or an antigen binding fragment thereof are provided. In some examples, instructions for using an anti-idiotype antibody to stimulate an input composition comprising cells expressing a chimeric antigen receptor comprising a SJ25C1 antibody or antigen binding fragment thereof are provided.
In some embodiments, instructions for using an anti-idiotype antibody to detect FMC63 antibodies or antigen binding fragments thereof, or chimeric antigen receptors comprising FMC63 antibodies or antigen binding fragments thereof, are provided, e.g., according to (or in conjunction with) any of the methods or assays. In some aspects, provided are instructions for using an anti-idiotype antibody to select or enrich for engineered cells from a population of cells that express a Chimeric Antigen Receptor (CAR) comprising antibody FMC63 or an antigen binding fragment thereof. In some embodiments, instructions for using an anti-idiotype antibody to stimulate an input composition comprising cells expressing a chimeric antigen receptor comprising an FMC63 antibody or antigen binding fragment thereof are provided.
In some embodiments, instructions for using the provided kits to detect molecules, such as antibodies, that bind to chimeric antigen receptors of cell therapies, e.g., antibodies generated by a humoral immune response to Chimeric Antigen Receptors (CARs), are provided. In some embodiments, instructions are provided for contacting a binding agent with a sample from a subject to which a cell therapy has been administered, the cell therapy comprising a cell engineered with a CAR comprising a target antibody that is an anti-CD 19 antibody (e.g., FMC63 or SJ25C 1) or an antigen binding fragment thereof, wherein the binding agent comprises an extracellular domain of the CAR comprising the target antibody or an antigen binding fragment thereof, or a portion of the extracellular domain. In some aspects, the specification further details detecting the presence or absence of a complex comprising a binding reagent and a molecule from a sample, the molecule binding to both the first and second binding reagents, optionally wherein the molecule is or comprises an antibody. In some aspects, the CD19 antibody is SJ25C1 or FMC63. In some other embodiments, instructions for using the binding reagent and the positive control sample are provided.
Definition of the definition
Unless defined otherwise, all terms of art, notations and other technical and scientific terms or words used herein are intended to have the same meaning as commonly understood by one of ordinary skill in the art to which the claimed subject matter pertains. In some cases, terms with commonly understood meanings are defined herein for clarity and/or for ease of reference, and the definitions contained herein should not be construed as indicating substantial differences from what is commonly understood in the art.
As used herein, the "corresponding form" of an antibody means that when comparing properties or activities of two antibodies, the properties are compared using the same form of antibody. For example, if it is stated that an antibody has a higher activity than the corresponding form of the first antibody, this means that a particular form of the antibody, such as an scFv, has a higher activity than the scFv form of the first antibody.
As used herein, recitation of a nucleotide or amino acid position "corresponding to" a nucleotide or amino acid position in a published sequence (such as listed in the sequence listing) refers to a nucleotide or amino acid position identified when aligned with the published sequence in order to maximize identity using standard alignment algorithms (such as the GAP algorithm). By aligning the sequences, the person skilled in the art can identify the corresponding residues, for example using conserved and identical amino acid residues as guide. Typically, to identify the corresponding positions, the amino acid sequences are aligned to obtain the highest level of matching (see, e.g., computational Molecular Biology, lesk, a.m., ed., oxford University Press, new York,1988;Biocomputing:Informatics and Genome Projects,Smith,D.W, ed., academic Press, new York,1993;Computer Analysis of Sequence Data,Part I,Griffin,A.M, and Griffin, h.g., eds, humana Press, new.Jersey,1994;Sequence Analysis in Molecular Biology,von Heinje,G, academic Press,1987, and Sequence Analysis Primer, gribskov, m.and Devereux, j., eds., M stock Press, new York,1991; carrilo et al (1988) SIAM J Applied Math 48:1073).
"effector functions" refer to those biological activities attributable to the Fc region of an antibody, which vary with the antibody isotype. Examples of antibody effector functions include: c1q binding and Complement Dependent Cytotoxicity (CDC); fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; down-regulation of cell surface receptors (e.g., B cell receptors); and B cell activation.
The term "Fc region" is used herein to define the C-terminal region of an immunoglobulin heavy chain that contains at least a portion of a constant region. The term includes native sequence Fc regions and variant Fc regions. In one embodiment, the human IgG heavy chain Fc region extends from Cys226 or from Pro230 to the carboxy terminus of the heavy chain. However, the C-terminal lysine (Lys 447) of the Fc region may or may not be present. Unless otherwise indicated herein, numbering of amino acid residues in the Fc region or constant region is according to the EU numbering system (also known as the EU index) as described by Kabat et al in Sequences of Proteins of Immunological Interest,5th Ed.Public Health Service,National Institutes of Health,Bethesda,MD,1991.
The terms "full length antibody", "whole antibody" and "whole antibody" are used interchangeably herein to refer to an antibody having a structure substantially similar to the structure of a natural antibody or having a heavy chain comprising an Fc region as defined herein.
An "isolated" antibody is an antibody that has been isolated from a component of its natural environment. In some embodiments, the antibodies are purified to greater than 95% or 99% purity as determined by, for example, electrophoresis (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis), or chromatography (e.g., ion exchange or reverse phase HPLC). For a review of methods of assessing antibody purity, see, e.g., flatman et al, J.chromatogrB 848:79-87 (2007).
An "isolated" nucleic acid refers to a nucleic acid molecule that has been isolated from a component of its natural environment. An isolated nucleic acid includes a nucleic acid molecule that is normally contained in a cell containing the nucleic acid molecule, but the nucleic acid molecule is present extrachromosomally or at a chromosomal location different from that of its natural chromosomal location.
"isolated nucleic acid encoding an anti-idiotype antibody" refers to one or more nucleic acid molecules encoding the heavy and light chains of the antibody (or fragments thereof), including such nucleic acid molecules in a single vector or in separate vectors, and such nucleic acid molecules are present at one or more locations in a host cell.
The terms "host cell", "host cell line" and "host cell culture" are used interchangeably and refer to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells. Host cells include "transformants" and "transformed cells" which include the primary transformed cell and the progeny derived therefrom, regardless of the number of passages. The offspring may not be exactly identical in nucleic acid content to the parent cell, but may contain mutations. Mutant offspring that have the same function or biological activity as screened or selected in the original transformed cell are included herein.
As used herein, "percent (%) amino acid sequence identity" and "percent identity" when used with respect to an amino acid sequence (reference polypeptide sequence) are defined as the percentage of amino acid residues in a candidate sequence (e.g., a subject antibody or fragment) that are identical to amino acid residues in the reference polypeptide sequence after sequence alignment and introduction of gaps (if desired, up to a maximum percent sequence identity, and without regard to any conservative substitutions as part of sequence identity). Alignment for determining the percent amino acid sequence identity can be accomplished in a variety of ways well known to those skilled in the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software. One skilled in the art can determine the appropriate parameters for aligning sequences, including any algorithms needed to achieve maximum alignment over the full length of the sequences compared.
Amino acid substitutions may include the replacement of one amino acid in a polypeptide with another amino acid. Substitutions may be conservative amino acid substitutions or non-conservative amino acid substitutions. Amino acid substitutions may be introduced into a binding molecule of interest (e.g., an antibody) and products selected for desired activity, such as retention/improved antigen binding, reduced immunogenicity, or improved ADCC or CDC.
Amino acids can generally be grouped according to the following common side chain properties:
(1) Hydrophobic: norleucine, met, ala, val, leu, ile;
(2) Neutral hydrophilic: cys, ser, thr, asn, gln;
(3) Acidic: asp, glu;
(4) Alkaline: his, lys, arg;
(5) Residues that affect chain orientation: gly, pro;
(6) Aromatic: trp, tyr, phe.
In some embodiments, conservative substitutions may involve swapping a member of one of these classes for another member of the same class. In some embodiments, non-conservative amino acid substitutions may involve exchanging members of one of these classes for another class.
The term "vector" as used herein refers to a nucleic acid molecule capable of transporting another nucleic acid to which it is linked. The term includes vectors that are self-replicating nucleic acid structures and that are incorporated into the genome of a host cell into which they have been introduced. Certain vectors are capable of directing the expression of nucleic acids to which they are operably linked. Such vectors are referred to herein as "expression vectors".
The term "package insert" is used to refer to instructions that are typically included in commercial packages of therapeutic products that contain information about the indication, usage, dosage, administration, combination therapy, contraindications and/or warnings of using such therapeutic products.
As used herein, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. For example, "a" or "an" means "at least one" or "one or more". It is to be understood that aspects and variations described herein include aspects and variations that "consist of and/or" consist essentially of.
Throughout this disclosure, various aspects of the claimed subject matter are presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as a rigid limitation on the scope of the claimed subject matter. Accordingly, the description of a range should be considered to specifically disclose all possible sub-ranges and individual values within the range. For example, where a range of values is provided, it is to be understood that each intervening value, to the extent stated, between the upper and lower limit of that range, and any other stated or intervening value in that stated range, is encompassed within the claimed subject matter. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also included in the claimed subject matter, subject to any specifically excluded limit in the stated range. Where a stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the claimed subject matter. This applies regardless of the width of the range.
The term "about" as used herein refers to the usual error range of the corresponding value as readily known to those skilled in the art. Reference herein to "about" a value or parameter includes (and describes) embodiments directed to that value or parameter itself. For example, a description referring to "about X" includes a description of "X".
The terms "polypeptide" and "protein" are used interchangeably and refer to a polymer of amino acid residues and are not limited to a minimum length. Polypeptides, including antibodies and antibody chains provided, as well as other peptides (e.g., linkers), may comprise amino acid residues including natural and/or unnatural amino acid residues. These terms also include post-expression modifications of the polypeptide, such as glycosylation, sialylation, acetylation, phosphorylation, and the like. In some aspects, the polypeptide may contain modifications relative to the natural or native sequence so long as the protein retains the desired activity. These modifications may be deliberate, such as by site-directed mutagenesis, or may be occasional, such as by mutation of the host producing the protein or by errors due to PCR amplification.
As used herein, a composition refers to any mixture of two or more products, substances or compounds, including cells. It may be a solution, suspension, liquid, powder, paste, aqueous, non-aqueous, or any combination thereof.
As used herein, a statement that a cell or cell population is "positive" for a particular marker refers to the detectable presence of the particular marker (typically a surface marker) on or in the cell. When referring to a surface marker, the term refers to the presence of surface expression detected by flow cytometry, e.g., by staining with an antibody that specifically binds to the marker and detecting the antibody, wherein staining is detectable by flow cytometry at a level substantially higher than that detected by the same procedure with an isotype-matched control under otherwise identical conditions and/or at a level substantially similar to that of cells known to be positive for the marker, and/or at a level substantially higher than that of cells known to be negative for the marker.
As used herein, a statement that a cell or cell population is "negative" for a particular marker refers to the absence of a substantial detectable presence of the particular marker (typically a surface marker) on or in the cell. When referring to a surface marker, the term refers to the lack of surface expression detected by flow cytometry, e.g., by staining with an antibody that specifically binds to the marker and detecting the antibody, wherein staining is not detected by flow cytometry at a level substantially higher than that detected by the same procedure with an isotype-matched control under otherwise identical conditions, and/or at a significantly lower level than cells known to be positive for the marker, and/or at a substantially similar level as cells known to be negative for the marker.
Exemplary embodiments
Wherein the embodiments provided herein are:
1. an anti-idiotype antibody or antigen-binding fragment thereof that specifically binds to a target antibody, which is antibody SJ25C1 or antigen-binding fragment thereof.
2. The anti-idiotype antibody or antigen-binding fragment of embodiment 1, wherein said antibody or antigen-binding fragment comprises:
a light chain Variable (VL) region having at least 90% sequence identity to the VL region amino acid sequence set forth in SEQ ID No. 5; and/or
A heavy chain Variable (VH) region having at least 90% sequence identity to the VH region amino acid sequence set forth in SEQ ID No. 1.
3. An antibody or antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment comprises:
a VL region having at least 90% sequence identity to the VL region amino acid sequence set forth in SEQ ID No. 5; and/or
A VH region having at least 90% sequence identity to the VH region amino acid sequence set forth in SEQ ID No. 1.
4. The anti-idiotype antibody or antigen-binding fragment of embodiment 2 or embodiment 3, wherein:
the VH region comprises heavy chain complementarity determining region 3 (CDR-H3) comprising the amino acid sequence set forth in SEQ ID NO. 11 or 84 or CDR-H3 comprised within the VH sequence set forth in SEQ ID NO. 1; and/or
The VL region comprises light chain complementarity determining region 3 (CDR-L3) comprising the amino acid sequence set forth in SEQ ID NO. 14 or 87 or comprises CDR-L3 comprised within the VL sequence set forth in SEQ ID NO. 5.
5. The anti-idiotype antibody or antigen-binding fragment of any one of embodiments 2-4, wherein:
the VH region comprises CDR-H1 and CDR-H2 comprising the amino acid sequences of CDR-H1 and CDR-H2 sequences, respectively, contained within the amino acid sequences of the VH region set out in SEQ ID NO 1; and/or
The VL region comprises CDR-L1 and CDR-L2, which comprise the amino acid sequences of CDR-L1 and CDR-L2 sequences, respectively, contained within the amino acid sequences of the VL region set forth in SEQ ID NO. 5.
6. The anti-idiotype antibody or antigen-binding fragment of any one of embodiments 2-5, wherein:
the VH region comprises CDR-H1 as set out in SEQ ID NO 9, 78, 79 or 80; CDR-H2 as set forth in SEQ ID NO 10, 81, 82 or 83; and CDR-H3 as set forth in SEQ ID NO 11 or 84; and/or
The VL region comprises the CDR-L1 set forth in SEQ ID NO 12 or 85; CDR-L2 as set forth in SEQ ID NO 13 or 86; and CDR-L3 as set forth in SEQ ID NO 14 or 87.
7. An anti-idiotype antibody, or antigen-binding fragment thereof, comprising:
CDR-H1, CDR-H2 and CDR-H3 comprising the amino acid sequences of CDR-H1, CDR-H2 and CDR-H3 sequences contained within the amino acid sequences of the VH region set forth in SEQ ID NO 1, respectively; and/or
CDR-L1, CDR-L2 and CDR-L3, which comprise the amino acid sequences of CDR-L1, CDR-L2 and CDR-L3 sequences, respectively, contained within the amino acid sequences of the VL region set forth in SEQ ID NO 5.
8. An anti-idiotype antibody, or antigen-binding fragment thereof, comprising:
CDR-H1 comprising the amino acid sequence SEQ ID NO 9, 78, 79 or 80; CDR-H2 comprising the amino acid sequence SEQ ID NO 10, 81, 82 or 83; and CDR-H3 comprising the amino acid sequence set forth in SEQ ID NO 11 or 84; and/or
CDR-L1 comprising the amino acid sequence SEQ ID NO 12 or 85; CDR-L2 comprising the amino acid sequence SEQ ID NO 13 or 86; and CDR-L3 comprising the amino acid sequence SEQ ID NO 14 or 87.
9. The anti-idiotype antibody or antigen-binding fragment thereof of any one of embodiments 1-8, wherein:
the VH region of the antibody or fragment comprises the amino acid sequence SEQ ID No. 1; and/or
The VL region of the antibody or fragment comprises the amino acid sequence SEQ ID NO. 5.
10. The anti-idiotype antibody of embodiment 9, or antigen-binding fragment thereof, wherein the VH region of said antibody or fragment comprises amino acid sequence SEQ ID No. 1, and the VL region of said antibody or fragment comprises amino acid sequence SEQ ID No. 5.
11. The anti-idiotype antibody or antigen-binding fragment of any of embodiments 1-10, wherein said target antibody or antigen-binding fragment comprises the heavy chain variable region set forth in SEQ ID NO. 23 and/or the light chain variable region set forth in SEQ ID NO. 24.
12. An anti-idiotype antibody or antigen-binding fragment thereof that specifically binds to a target antibody, which is antibody FMC63 or antigen-binding fragment thereof.
13. The anti-idiotype antibody or antigen-binding fragment of embodiment 12, wherein said antibody or antigen-binding fragment comprises:
a light chain Variable (VL) region having at least 90% sequence identity to the VL region amino acid sequences set forth in SEQ ID NOs 40 or 62; and/or
A heavy chain Variable (VH) region having at least 90% sequence identity to the VH region amino acid sequence set forth in SEQ ID No. 36 or 58.
14. An antibody or antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment comprises:
a VL region having at least 90% sequence identity to the VL region amino acid sequence set forth in SEQ ID NO. 40 or 62; and/or
A VH region having at least 90% sequence identity to the VH region amino acid sequence set forth in SEQ ID No. 36 or 58.
15. The anti-idiotype antibody or antigen-binding fragment of any one of embodiments 13-14, wherein:
The VH region comprises:
comprising the amino acid sequence GYX 3 FX 5 X 6 YX 8 MX 10 Heavy chain complementarity determining region 1 (CDR-H1) (SEQ ID NO: 108), wherein X 3 Is T or S, X 5 Is T or S, X 6 Is D or R, X 8 Is Y or W, and X 10 Is K or N;
comprising the amino acid sequence WIGX 4 IX 6 PX 8 X 9 X 10 X 11 TX 13 X 14 NQX 17 FKX 20 Heavy chain complementarity determining region 2 (CDR-H2) of (SEQ ID NO: 109), wherein X 4 Is D or M, X 6 Is N or H, X 8 Is N or S, X 9 Is N or D, X 10 Is G or S, X 11 Is G or E, X 13 Is D or R, X 14 Y or L, X 17 Is N or K, and X 20 Is G or D;
comprising the amino acid sequence AX 2 X 3 X 4 X 5 X 6 X 7 X 8 X 9 X 10 X 11 X 12 X 13 X 14 X 15 Heavy chain complementarity determining region 3 (CDR-H3) of (SEQ ID NO: 110), wherein X 2 Is R or S, X 3 Is E or I, X 4 Is G or Y, X 5 Is N or Y, X 6 Is N or E, X 7 Is Y or null, X 8 Is G or null, X 9 Is S or null, X 10 R is null or null, X 11 Is D or null, X 12 Is A or null value, X 13 Is M or null, X 14 Is D or E, and X 15 Y or A; and/or
The VL region comprises:
comprising the amino acid sequence X 1 AX 3 X 4 X 5 X 6 X 7 X 8 YX 10 X 11 Light chain complementarity determining region 1 (CDR-L1) of WY (SEQ ID NO: 111), wherein X 1 Is S or R, X 3 Is S or R, X 4 Is S or G, X 5 Is G or N, X 6 Is V or I, X 7 Is I or H, X 8 Is N or null, X 10 Is M or L, and X 11 Y or A;
comprising the amino acid sequence X 1 X 2 X 3 YX 5 X 6 X 7 X 8 LAX 11 Light chain complementarity determining region 2 (CDR-L2) of (SEQ ID NO: 112), wherein X 1 Is P or L, X 2 Is W or L, X 3 Is I or V, X 5 Is L or N, X 6 Is T or A, X 7 Is S or K, X 8 Is N or T, and X 11 Is S or D;
comprising the amino acid sequence QX 2 X 3 X 4 X 5 X 6 PX 8 T (SEQ ID NO: 113) light chain complementarity determining region 3 (CDR-L3), wherein X 2 Is Q or H, X 3 Is W or F, X 4 Is S or W, X 5 Is S or W, X 6 Is N or T, and X 8 Is L or Y.
16. The anti-idiotype antibody or antigen-binding fragment of embodiment 15, wherein:
the complementarity determining region 3 (CDR-H3) comprises the amino acid sequence set forth in SEQ ID NO. 94 or 104 or comprises CDR-H3 contained within the VH sequence set forth in SEQ ID NO. 36 or 58; and/or
The light chain complementarity determining region 3 (CDR-L3) comprises the amino acid sequence set forth in SEQ ID NO. 97 or 107 or comprises CDR-L3 contained within the VL sequence set forth in SEQ ID NO. 40 or 62.
17. The anti-idiotype antibody or antigen-binding fragment of any one of embodiments 13-16, wherein:
the VH region comprises CDR-H1 and CDR-H2 comprising the amino acid sequences of CDR-H1 and CDR-H2 sequences contained within the amino acid sequences of the VH region set out in SEQ ID NO 36 or 58, respectively; and/or
The VL region comprises CDR-L1 and CDR-L2, which comprise the amino acid sequences of CDR-L1 and CDR-L2 sequences, respectively, contained within the amino acid sequences of the VL region set forth in SEQ ID NOS 40 or 62.
18. The anti-idiotype antibody or antigen-binding fragment of any one of embodiments 13-17, wherein:
the VH region comprises CDR-H1 as set out in SEQ ID NO 88, 89, 90, 98, 99 or 100; CDR-H2 as set forth in SEQ ID NO 91, 92, 93, 101, 102 or 103; and CDR-H3 as set forth in SEQ ID NO 94 or 104; and/or
The VL region comprises CDR-L1 as set forth in SEQ ID NO 95 or 105; CDR-L2 as set forth in SEQ ID NO 96 or 106; and CDR-L3 as set forth in SEQ ID NO 97 or 107.
19. An anti-idiotype antibody, or antigen-binding fragment thereof, comprising:
CDR-H1, CDR-H2 and CDR-H3 comprising the amino acid sequences of CDR-H1, CDR-H2 and CDR-H3 sequences contained within the amino acid sequences of the VH regions set forth in SEQ ID NO 36 or 58, respectively; and/or
CDR-L1, CDR-L2 and CDR-L3, comprising the amino acid sequences of CDR-L1, CDR-L2 and CDR-L3 sequences contained within the amino acid sequences of the VL region set forth in SEQ ID NO. 40 or 62, respectively.
20. An anti-idiotype antibody, or antigen-binding fragment thereof, comprising:
CDR-H1 comprising the amino acid sequence SEQ ID NO 88, 89, 90, 98, 99 or 100; CDR-H2 comprising the amino acid sequence SEQ ID NO 91, 92, 93, 101, 102 or 103; and CDR-H3 comprising the amino acid sequence set forth in SEQ ID NO 94 or 104; and/or
CDR-L1 comprising the amino acid sequence SEQ ID NO 95 or 105; CDR-L2 comprising the amino acid sequence SEQ ID NO 96 or 106; and CDR-L3 comprising the amino acid sequence SEQ ID NO 97 or 107.
21. The anti-idiotype antibody or antigen-binding fragment thereof of any one of embodiments 13-21, wherein:
the VH region of the antibody or fragment comprises the amino acid sequence SEQ ID No. 36 or 58; and/or
The VL region of the antibody or fragment comprises the amino acid sequence SEQ ID NO. 40 or 62.
22. The anti-idiotype antibody of embodiment 21, or antigen-binding fragment thereof, wherein the VH region of said antibody or fragment comprises amino acid sequence SEQ ID No. 36 or 58, and the VL region of said antibody or fragment comprises amino acid sequence SEQ ID No. 40 or 62.
23. The anti-idiotype antibody or antigen-binding fragment of any one of embodiments 13-22, wherein:
the VH region comprises CDR-H1 as set out in SEQ ID NO 44, 88, 89 or 90; CDR-H2 as set forth in SEQ ID NO 45, 91, 92 or 93; and CDR-H3 as set forth in SEQ ID NO 46 or 94; and/or
The VL region comprises CDR-L1 as set forth in SEQ ID NO 47 or 95; CDR-L2 as set forth in SEQ ID NO 48 or 96; and CDR-L3 as set forth in SEQ ID NO 49 or 97.
24. The anti-idiotype antibody or antigen-binding fragment of any one of embodiments 13-23, wherein:
the VH region comprises CDR-H1 as set out in SEQ ID NO 65, 98, 99 or 100; CDR-H2 as set forth in SEQ ID NO 66, 101, 102 or 103; and CDR-H3 as set forth in SEQ ID NO 67 or 104; and/or
The VL region comprises the CDR-L1 set forth in SEQ ID NO 68 or 105; CDR-L2 as set forth in SEQ ID NO 69 or 106; and CDR-L3 as set forth in SEQ ID NO. 100 or 107.
25. The anti-idiotype antibody or antigen-binding fragment of any one of embodiments 13-24, wherein:
the VH region comprises CDR-H1, CDR-H2 and CDR-H3 comprising the amino acid sequences of CDR-H1, CDR-H2 and CDR-H3 contained within the amino acid sequences of the VH region set out in SEQ ID NO 36; and/or
The VL region comprises CDR-L1, CDR-L2 and CDR-L3, which comprise the amino acid sequences of CDR-L1, CDR-L2 and CDR-L3 sequences, respectively, contained within the amino acid sequences of the VL region set forth in SEQ ID NO. 40.
26. The anti-idiotype antibody or antigen-binding fragment of any one of embodiments 13-24, wherein:
the VH region comprises CDR-H1, CDR-H2 and CDR-H3 comprising the amino acid sequences of CDR-H1, CDR-H2 and CDR-H3 contained within the amino acid sequence of the VH region set out in SEQ ID NO 58; and/or
The VL region comprises CDR-L1, CDR-L2 and CDR-L3, which comprise the amino acid sequences of CDR-L1, CDR-L2 and CDR-L3 sequences, respectively, contained within the amino acid sequences of the VL region set forth in SEQ ID NO. 62.
27. The anti-idiotype antibody or antigen-binding fragment of any one of embodiments 13-25, wherein:
the VH region of the antibody or fragment comprises the amino acid sequence SEQ ID No. 36; and/or
The VL region of the antibody or fragment comprises the amino acid sequence SEQ ID NO. 40.
28. The anti-idiotype antibody or antigen-binding fragment of any one of embodiments 13-24 and 26, wherein:
the VH region of the antibody or fragment comprises the amino acid sequence SEQ ID NO 58; and/or
The VL region of the antibody or fragment comprises the amino acid sequence SEQ ID NO. 62.
29. The anti-idiotype antibody or antigen-binding fragment of any of embodiments 1-28, wherein said target antibody or antigen-binding fragment is a single-chain fragment.
30. The anti-idiotype antibody or antigen-binding fragment of embodiment 29, wherein said fragment comprises an antibody variable region linked by a flexible linker.
31. The anti-idiotype antibody or antigen-binding fragment of embodiment 29 or embodiment 30, wherein the fragment comprises an scFv.
32. The anti-idiotype antibody or antigen-binding fragment of any one of embodiments 1-11 and 29-31, wherein said target antibody or antigen-binding fragment:
Comprising the heavy chain variable region set forth in SEQ ID NO. 23 and/or the light chain variable region set forth in SEQ ID NO. 24; and/or
Is an scFv comprising the amino acid sequence set forth in SEQ ID NO. 28.
33. The anti-idiotype antibody or antigen-binding fragment of any one of embodiments 12-31, wherein said target antibody or antigen-binding fragment:
comprising the heavy chain variable region set forth in SEQ ID NO. 30 and/or the light chain variable region set forth in SEQ ID NO. 31; and/or
Is an scFv comprising the amino acid sequence set forth in SEQ ID NO 34.
34. An anti-idiotype antibody or antigen-binding fragment thereof, wherein said anti-idiotype antibody or antigen-binding fragment specifically binds to an epitope of a target antibody or antigen-binding fragment thereof that is the same as or overlaps with an epitope specifically bound by the anti-idiotype antibody or antigen-binding fragment of any one of embodiments 1-33.
35. The anti-idiotype antibody or antigen-binding fragment of any one of embodiments 1-34, wherein:
the target antibody or antigen binding fragment is within or contained within an antigen binding domain of an extracellular portion of a Chimeric Antigen Receptor (CAR); and/or
The anti-idiotype antibody or antigen-binding fragment specifically binds to a target antibody or antigen-binding fragment contained within or within the antigen-binding domain of the extracellular portion of the CAR.
36. The anti-idiotype antibody or antigen-binding fragment of embodiment 35, wherein the target antibody or antigen-binding fragment is an scFv and the anti-idiotype antibody or antigen-binding fragment specifically binds to an epitope in the scFv of the CAR.
37. The anti-idiotype antibody of any of embodiments 1-11, 29-32, and 35, or an antigen-binding fragment thereof, wherein said antibody or fragment specifically binds to a single chain variable fragment (scFv) derived from antibody SJ25C1 comprised in the extracellular portion of a chimeric antigen receptor, optionally wherein the scFv derived from antibody SJ25C1 comprises the heavy chain variable region set forth in SEQ ID No. 23 and/or the light chain variable region set forth in SEQ ID No. 24; and/or comprises the amino acid sequence set forth in SEQ ID NO. 28.
38. The anti-idiotype antibody or antigen-binding fragment of any of embodiments 12-31 and 33-35, wherein said antibody or fragment specifically binds to a single chain variable fragment (scFv) derived from antibody FMC63 comprised in the extracellular portion of a chimeric antigen receptor, optionally wherein the scFv derived from antibody FMC63 comprises the heavy chain variable region set forth in SEQ ID No. 30 and/or the light chain variable region set forth in SEQ ID No. 31; and/or comprises the amino acid sequence set forth in SEQ ID NO. 34. 39. The anti-idiotype antibody or antigen-binding fragment of any of embodiments 1-38, wherein said anti-idiotype antibody or antigen-binding fragment specifically binds to an epitope within or comprising all or part of the Complementarity Determining Regions (CDRs) of the target antibody or antigen-binding fragment.
40. The anti-idiotype antibody or antigen-binding fragment of any of embodiments 35-39, wherein the CAR further comprises a transmembrane domain linked to the antigen-binding domain by a spacer.
41. The anti-idiotype antibody of embodiment 40, wherein said spacer comprises an extracellular portion from CD28, said CD28 optionally being human CD28.
42. The anti-idiotype antibody or antigen-binding fragment of embodiment 41, wherein the extracellular portion from CD28 comprises the amino acid sequence set forth in SEQ ID NO. 27.
43. The anti-idiotype antibody or antigen-binding fragment of any of embodiments 40-42, wherein said transmembrane domain comprises a transmembrane portion of CD28, said CD28 optionally being human CD28.
44. The anti-idiotype antibody or antigen binding fragment of any of embodiments 40-43, wherein said antibody or fragment does not bind to an epitope in the spacer domain of the CAR.
45. The anti-idiotype antibody or antigen-binding fragment of any of embodiments 1-44, wherein said antibody or fragment does not bind or does not specifically bind to CD28 or a portion thereof, said CD28 optionally being human CD28, optionally comprising an extracellular portion of CD28, optionally comprising the amino acid sequence set forth in SEQ ID No. 27.
46. The anti-idiotype antibody or antigen-binding fragment of any of embodiments 1-45, wherein said antibody or fragment does not bind to an epitope in an Fc domain, optionally a human IgG1Fc domain.
47. The anti-idiotype antibody or antigen-binding fragment of any of embodiments 1-46, wherein said target antibody or antigen-binding fragment specifically binds human CD 19.
48. The anti-idiotype antibody or antigen-binding fragment thereof of any of embodiments 1-47, wherein said anti-idiotype antibody or fragment does not cross-react with another anti-CD 19 antibody, said anti-CD 19 antibody optionally being comprised in the extracellular antigen-binding domain of another CAR.
49. The anti-idiotype antibody or antigen-binding fragment thereof of any of embodiments 1-48, wherein said anti-idiotype antibody or fragment does not cross-react with another CAR.
50. The anti-idiotype antibody or antigen-binding fragment thereof of any of embodiments 1-49, wherein said anti-idiotype antibody or fragment is an agonist antibody to a CAR comprising the target antibody or antigen-binding fragment.
51. The anti-idiotype antibody or antigen-binding fragment thereof of any of embodiments 1-50, wherein said antibody or fragment is an antagonist of a CAR comprising the target antibody or antigen-binding fragment.
52. The anti-idiotype antibody of any one of embodiments 1-51, or antigen-binding fragment thereof, that is humanized.
53. The anti-idiotype antibody or antigen-binding fragment thereof of any of embodiments 1-52, which is recombinant.
54. The anti-idiotype antibody of any of embodiments 1-53, or an antigen-binding fragment thereof, which is monoclonal.
55. The anti-idiotype antibody of any of embodiments 1-54, or an antigen-binding fragment thereof, which is an antigen-binding fragment.
56. The anti-idiotype antibody or antigen-binding fragment of embodiment 55, wherein said antigen-binding fragment is selected from the group consisting of a fragment antigen-binding (Fab) fragment, F (ab') 2 Fragments, fab' fragments, fv fragments, single chain variable fragments (scFv), or single domain antibodies.
57. The anti-idiotype antibody or antigen-binding fragment of any of embodiments 1-54, comprising at least a portion of an immunoglobulin constant region.
58. The anti-idiotype antibody or antigen-binding fragment of embodiment 57, wherein at least a portion of the immunoglobulin constant region comprises an Fc region or a portion of an Fc comprising CH2 and CH3 domains.
59. The anti-idiotype antibody or antigen-binding fragment of embodiment 57 or embodiment 58, wherein said constant region is derived from human IgG.
60. The anti-idiotype antibody or antigen-binding fragment of any of embodiments 57-59, which is an intact antibody or a full-length antibody.
61. A conjugate comprising an anti-idiotype antibody or antigen-binding fragment of any of embodiments 1-60 and a heterologous molecule or moiety.
62. The conjugate of embodiment 61, wherein the heterologous molecule or moiety is a label.
63. The conjugate of embodiment 62, wherein the label is selected from the group consisting of a fluorescent dye, a fluorescent protein, a radioisotope, a chromophore, a metal ion, a gold particle, a silver particle, a magnetic particle, a polypeptide, an enzyme, streptavidin, biotin, a luminescent compound, or an oligonucleotide.
64. The conjugate of embodiment 62, wherein said heterologous molecule or moiety is a protein, peptide, nucleic acid or small molecule, which is optionally or comprises a toxin Strep-Tag.
65. A nucleic acid molecule encoding the heavy and/or light chain of the anti-idiotype antibody of any of embodiments 1-60 or an antigen binding fragment thereof.
66. The nucleic acid molecule of embodiment 65 comprising:
a nucleotide sequence set forth in SEQ ID NO. 15 encoding (i) a heavy chain variable region, (ii) a nucleotide sequence having at least 90% sequence identity to the nucleotide sequence set forth in SEQ ID NO. 15; or (iii) a degenerate sequence of (i) or (ii); and/or
A nucleotide sequence set forth in SEQ ID NO. 19 encoding (iv) a light chain variable region, (v) a nucleotide sequence having at least 90% sequence identity to the nucleotide sequence set forth in SEQ ID NO. 19; or (vi) a degenerate sequence of (iv) or (v).
67. The nucleic acid molecule of embodiment 65 or embodiment 66 comprising:
a nucleotide sequence set forth in SEQ ID NO. 17 encoding (i) a heavy chain, (ii) a nucleotide sequence having at least 90% sequence identity to the nucleotide sequence set forth in SEQ ID NO. 17; or (iii) a degenerate sequence of (i) or (ii); and/or
A nucleotide sequence set forth in SEQ ID NO. 21 encoding (iv) a light chain, (v) a nucleotide sequence having at least 90% sequence identity to the nucleotide sequence set forth in SEQ ID NO. 21; or (vi) a degenerate sequence of (iv) or (v).
68. The nucleic acid molecule of embodiment 65 comprising:
a nucleotide sequence set forth in SEQ ID NO. 50 or 71 encoding (i) a heavy chain variable region, (ii) a nucleotide sequence having at least 90% sequence identity to the nucleotide sequence set forth in SEQ ID NO. 50 or 71; or (iii) a degenerate sequence of (i) or (ii); and/or
A nucleotide sequence set forth in SEQ ID NO. 54 or 75 encoding (iv) a light chain variable region, (v) a nucleotide sequence having at least 90% sequence identity to the nucleotide sequence set forth in SEQ ID NO. 54 or 75; or (vi) a degenerate sequence of (iv) or (v).
69. The nucleic acid molecule of embodiment 65 or embodiment 68 comprising:
a nucleotide sequence set forth in SEQ ID NO. 52 or 73 encoding (i) a heavy chain, (ii) a nucleotide sequence having at least 90% sequence identity to the nucleotide sequence set forth in SEQ ID NO. 52 or 73; or (iii) a degenerate sequence of (i) or (ii); and/or
A nucleotide sequence set forth in SEQ ID NO. 56 or 76 encoding (iv) a light chain, (v) a nucleotide sequence having at least 90% sequence identity to the nucleotide sequence set forth in SEQ ID NO. 56 or 76; or (vi) a degenerate sequence of (iv) or (v).
70. The nucleic acid molecule of any one of embodiments 65-69, wherein the nucleotide sequence encoding the heavy chain and/or the light chain comprises a signal sequence.
71. A vector comprising the nucleic acid molecule of any one of embodiments 65-70.
72. A cell comprising the anti-idiotype antibody of any of embodiments 1-41 or an antigen-binding fragment thereof or the nucleic acid molecule of any of embodiments 65-70.
73. A method of producing an anti-idiotype antibody or antigen-binding fragment thereof, comprising expressing the heavy and/or light chain encoded by the nucleic acid molecule of any of embodiments 65-70 or the vector of embodiment 71 in a suitable host cell, and recovering or isolating the antibody.
74. A method of producing an anti-idiotype antibody or antigen-binding fragment thereof, comprising culturing the cells of embodiment 72 under conditions that express heavy and/or light chains, and recovering or isolating the antibody.
75. An anti-idiotype antibody, or antigen-binding fragment thereof, produced by the method of embodiment 73 or embodiment 74.
76. A composition comprising the anti-idiotype antibody of any of embodiments 1-60, or an antigen-binding fragment thereof, the conjugate of any of embodiments 61-64, or the cell of embodiment 72.
77. The composition of embodiment 76, further comprising a pharmaceutically acceptable excipient.
78. A kit comprising one or more of the following: the anti-idiotype antibody or antigen-binding fragment thereof of any of embodiments 1-60, the conjugate of any of embodiments 61-64, the nucleic acid of any of embodiments 65-70, and optionally instructions for use.
79. The kit of embodiment 78, further comprising a reagent or support for immobilizing an anti-idiotype antibody or antigen-binding fragment or conjugate thereof, wherein the reagent or support is a bead, column, microwell, stick, filter, strip, or soluble oligomeric streptavidin mutein reagent.
80. A method of detecting a target antibody or antigen-binding fragment thereof, comprising:
(a) Contacting a composition comprising a target antibody (which is antibody SJ25C1 or antigen binding fragment) with the anti-idiotype antibody of any of embodiments 1-11 and 29-32, 34-37 and 39-60 or the antigen binding fragment thereof, or the conjugate of any of embodiments 61-64, which specifically binds to the target antibody (which is antibody SJ25C1 or antigen binding fragment thereof); and
(b) Anti-idiotype antibodies that bind to the target antibody or antigen-binding fragment are detected.
81. A method of detecting a target antibody or antigen-binding fragment thereof, comprising:
(a) Contacting a composition comprising a target antibody (which is antibody FMC63 or antigen binding fragment thereof) with the anti-idiotype antibody of any of embodiments 12-31, 33-36, and 38-60, or the antigen binding fragment thereof, or the conjugate of any of embodiments 61-64, which specifically binds to the target antibody (which is antibody FMC63 or antigen binding fragment thereof); and
(b) Anti-idiotype antibodies that bind to the target antibody or antigen-binding fragment are detected.
82. The method of embodiment 81, wherein the target antibody or antigen binding fragment binds to or is expressed on the surface of a cell, and the detecting in (b) comprises detecting a cell that binds to an anti-idiotype antibody.
83. The method of embodiment 82, wherein the cell expresses a CAR comprising the target antibody or antigen-binding fragment on its surface.
84. A method of detecting a CAR comprising a target antibody or antigen-binding fragment thereof, comprising:
(a) Contacting a cell expressing a Chimeric Antigen Receptor (CAR) comprising a target antibody (which is antibody SJ25C1 or antigen binding fragment thereof) with the anti-idiotype antibody of any one of embodiments 1-11 and 29-32, 34-37 and 39-60 or the antigen binding fragment thereof, or the conjugate of any one of embodiments 61-64, which specifically binds to the target antibody (which is antibody SJ25C1 or antigen binding fragment thereof); and
(b) Cells bound to the anti-idiotype antibody are detected.
85. A method of detecting a CAR comprising a target antibody or antigen-binding fragment thereof, comprising:
(a) Contacting a cell expressing a Chimeric Antigen Receptor (CAR) comprising a target antibody that is antibody FMC63 or an antigen binding fragment thereof with an anti-idiotype antibody of any one of embodiments 12-31, 33-36, and 38-60 or an antigen binding fragment thereof, or a conjugate of any one of embodiments 61-64, which specifically binds to the target antibody that is antibody FMC63 or an antigen binding fragment thereof; and
(b) Cells bound to the anti-idiotype antibody are detected.
86. The method of any one of embodiments 80-85, wherein the anti-idiotype antibody or antigen-binding fragment thereof is labeled directly or indirectly for detection.
87. A method of selecting cells from a population of cells, comprising:
(a) Contacting a population of cells expressing a Chimeric Antigen Receptor (CAR) comprising or bound to a target antibody with an anti-idiotype antibody of any of embodiments 1-11 and 29-32, 34-37 and 39-60, or an antigen-binding fragment thereof, or a conjugate of any of embodiments 61-64, which specifically binds to the target antibody (which is antibody SJ25C1 or an antigen-binding fragment thereof), wherein the target antibody is antibody SJ25C1 or an antigen-binding fragment thereof; and
(b) Cells that bind to the anti-idiotype antibody are selected.
88. A method of selecting cells from a population of cells, comprising:
(a) Contacting a population of cells expressing a Chimeric Antigen Receptor (CAR) comprising or binding to a target antibody with an anti-idiotype antibody of any one of embodiments 12-31, 33-36, and 38-60, or an antigen-binding fragment thereof, or a conjugate of any one of embodiments 61-64, which specifically binds to the target antibody (which is antibody FMC63 or an antigen-binding fragment thereof), wherein the target antibody is antibody FMC63 or an antigen-binding fragment thereof; and
(b) Cells that bind to the anti-idiotype antibody are selected.
89. The method of embodiment 87 or embodiment 88, wherein the cells that bind to the anti-idiotype antibody are selected by affinity-based separation.
90. The method of embodiment 89, wherein the affinity-based separation is an immunoaffinity-based separation.
91. The method of embodiment 89 or embodiment 90, wherein the affinity-based separation is accomplished by flow cytometry.
92. The method of embodiment 89 or embodiment 90, wherein the affinity-based separation is accomplished by magnetically activated cell sorting. 93. The method of embodiment 89 or embodiment 90, wherein the affinity-based separation comprises affinity chromatography. 94. The method of embodiment 92 or embodiment 93, wherein the anti-idiotype antibody is reversibly bound or immobilized on a support or stationary phase.
95. A method of stimulating cells comprising incubating an input composition of cells expressing a Chimeric Antigen Receptor (CAR) comprising a target antibody (which is antibody SJ25C1 or antigen binding fragment thereof) with an anti-idiotype antibody of any of embodiments 1-11 and 29-32, 34-37 and 39-60 or an antigen binding fragment thereof, or a conjugate of any of embodiments 61-64, which specifically binds to the target antibody (which is antibody SJ25C1 or antigen binding fragment thereof), thereby generating an output composition comprising stimulated cells.
96. A method of stimulating cells comprising incubating an input composition of cells expressing a Chimeric Antigen Receptor (CAR) comprising a target antibody (which is antibody FMC63 or antigen binding fragment thereof) with an anti-idiotype antibody or antigen binding fragment thereof of any of embodiments 12-31, 33-36, and 38-60, or a conjugate of any of embodiments 61-64, which specifically binds to the target antibody (which is antibody FMC63 or antigen binding fragment thereof), thereby generating an output composition comprising stimulated cells.
97. A method of producing a cellular composition comprising:
(a) Introducing a nucleic acid molecule encoding a Chimeric Antigen Receptor (CAR) into a cell, thereby generating an input composition; and
(b) Incubating the input composition with an anti-idiotype antibody or antigen-binding fragment thereof specific for an antigen receptor CAR, thereby producing a cellular composition.
98. The method of embodiment 97, wherein the CAR comprises a target antibody that specifically binds to CD 19.
99. The method of embodiment 98, wherein the target antibody is antibody SJ25C1 or an antigen binding fragment thereof.
100. The method of any one of embodiments 97-99, wherein the anti-idiotype antibody or antigen-binding fragment thereof is the anti-idiotype antibody or antigen-binding fragment of any one of embodiments 1-11 and 29-32, 34-37, and 39-60, which specifically binds to a target antibody (which is antibody SJ25C1 or antigen-binding fragment thereof).
101. The method of embodiment 98, wherein the target antibody is antibody FMC63 or an antigen binding fragment thereof.
102. The method of any one of embodiments 97-98 and 101, wherein the anti-idiotype antibody or antigen-binding fragment thereof that specifically binds to a target antibody (which is antibody FMC63 or antigen-binding fragment thereof) that is antibody FMC63 of any one of embodiments 12-31, 33-36 and 38-60.
103. The method of any one of embodiments 97-102, wherein the introducing in (a) comprises introducing the nucleic acid molecule into the cell by viral transduction, transposition, electroporation, or chemical transfection.
104. The method of any one of embodiments 97-103, wherein the introducing in (a) comprises introducing the nucleic acid molecule into the cell by transduction with a viral vector comprising the nucleic acid molecule, optionally wherein the viral vector is a retroviral vector or a lentiviral vector.
105. The method of any one of embodiments 97-103, wherein the introducing in (a) comprises introducing the nucleic acid molecule into the cell by transposition with a transposon comprising the nucleic acid molecule.
106. The method of any one of embodiments 97-103, wherein the introducing in (a) comprises introducing the nucleic acid molecule into the cell by electroporation or transfection of a vector comprising the nucleic acid molecule.
107. The method of any one of embodiments 97-106, further comprising the step of activating the cells prior to step (a).
108. The method of embodiment 107, wherein the step of activating the cell comprises contacting the cell with a CD3 agonist and optionally a CD28 agonist.
109. The method of embodiment 108, wherein the step of activating the cells comprises contacting the cells with an agent comprising an agonist anti-CD 3 antibody and an anti-CD 28 antibody.
110. The method of any one of embodiments 95-109, wherein the incubation is performed under conditions in which the anti-idiotype antibody or antigen-binding fragment thereof binds to the CAR, thereby inducing or modulating a signal in one or more cells of the input composition.
111. The method of any one of embodiments 95-110, wherein the cell comprises a T cell.
112. The method of embodiment 111, wherein the T cells comprise cd4+ and/or cd8+ T cells.
113. The method of any one of embodiments 95-112, wherein the anti-idiotype antibody or antigen-binding fragment thereof is immobilized to a solid support, optionally comprising or conjugated to an agent comprising a plurality of binding sites capable of reversibly binding to said anti-idiotype antibody or antigen-binding fragment thereof.
114. The method of any one of embodiments 95-112, wherein the anti-idiotype antibody or antigen-binding fragment thereof is immobilized to a soluble agent, optionally or comprising a plurality of binding sites capable of reversibly binding to said anti-idiotype antibody or antigen-binding fragment thereof.
115. The method of embodiment 113 or embodiment 114, wherein the agent comprises a streptavidin mutein.
116. The method of any of embodiments 95-115, wherein incubating is at least or about at least 5 minutes, 10 minutes, 30 minutes, 60 minutes, 2 hours, 6 hours, 12 hours, 24 hours, 36 hours, 48 hours, 72 hours, or 96 hours.
117. The method of any of embodiments 95-116, wherein the input composition comprises less than or less than about 60%, less than or less than about 50%, less than or less than about 40%, less than or less than about 30%, less than or less than about 20%, or less than or about 10% of CAR-expressing cells, by percent of total cells in the composition.
118. The method of any one of embodiments 95-117, wherein:
the number of CAR-expressing cells in the output composition is increased by greater than 1.2-fold, 1.5-fold, 2.0-fold, 3.0-fold, 4.0-fold, 5.0-fold, 10-fold, or more as compared to the number of CAR-expressing cells in the input composition; and/or
The percentage of CAR-expressing cells in the output composition is increased by greater than 10%, 20%, 40%, 50%, 60%, 70%, 80% or more as compared to the total cells in the composition.
119. The method of any one of embodiments 95-118, wherein the CAR-expressing cells are not selected or enriched for the cells prior to introduction and/or incubation.
120. The method of any one of embodiments 80, 82-84, 86, 87, 89-95, 97-100, 103-119, wherein said target antibody or antigen binding fragment comprises the heavy chain variable region set forth in SEQ ID No. 23 and/or the light chain variable region set forth in SEQ ID No. 24.
121. The method of any one of embodiments 81, 82, 83, 85, 86, 88-94, 96-99, 101 and 102-119, wherein said target antibody or antigen binding fragment comprises the heavy chain variable region set forth in SEQ ID No. 30 and/or the light chain variable region set forth in SEQ ID No. 31.
122. A method of purifying an antibody or antigen-binding fragment thereof, comprising:
(a) Contacting a composition comprising a target antibody (which is antibody SJ25C1 or antigen binding fragment thereof) with the anti-idiotype antibody of any of embodiments 1-11 and 29-32, 34-37 and 39-60 or antigen binding fragment thereof, or the conjugate of any of embodiments 61-64, which specifically binds to the target antibody (which is antibody SJ25C1 or antigen binding fragment thereof); and
(b) Isolating the complex comprising the anti-idiotype antibody.
123. A method of purifying an antibody or antigen-binding fragment thereof, comprising:
(a) Contacting a composition comprising a target antibody (which is antibody FMC63 or antigen binding fragment thereof) with the anti-idiotype antibody of any of embodiments 12-31, 33-36, and 38-60 or the antigen binding fragment thereof, or the conjugate of any of embodiments 61-64, which specifically binds to the target antibody (which is antibody FMC63 or antigen binding fragment thereof); and
(b) Isolating the complex comprising the anti-idiotype antibody.
124. The method of embodiment 122 or embodiment 123, wherein the complex comprising the anti-idiotype antibody is isolated by affinity-based separation.
125. The method of embodiment 124, wherein the affinity-based separation is an immunoaffinity-based separation.
126. The method of embodiment 124, wherein the affinity-based separation is magnetic-based separation.
127. The method of embodiment 124, wherein the affinity-based separation comprises affinity chromatography.
128. A method of identifying an anti-idiotype antibody or antigen-binding fragment comprising:
(a) Introducing a soluble immunoreagent comprising an antigen-binding fragment of a target antibody fused to a solubilising moiety into a subject; and
(b) An antibody from the subject that specifically binds to the target antibody or antigen-binding fragment thereof is identified.
129. The method of embodiment 128, wherein the antigen binding fragment comprises a variable heavy chain region and/or a variable light chain region of the target antibody.
130. The method of embodiment 128 or embodiment 129, wherein the antigen binding fragment is a single-stranded fragment.
131. The method of embodiment 130, wherein the antigen binding fragment is an scFv.
132. The method of any one of embodiments 128-131, wherein the antigen binding fragment is within or contained within an antigen binding domain of an extracellular portion of a Chimeric Antigen Receptor (CAR).
133. The method of any one of embodiments 128-132, wherein the solubilising moiety is an Fc domain or fragment thereof, optionally a human IgG1Fc.
134. The method of embodiment 133, wherein said solubilizing module is an Fc domain lacking a hinge region.
135. The method of embodiment 134, wherein said solubilizing module comprises the amino acid sequence set forth in SEQ ID NO. 32.
136. The method of any one of embodiments 128-135, wherein identifying the antibody comprises:
(i) Isolating B cells from the spleen of the subject and fusing them with immortalized B cells to produce hybridomas;
(ii) Screening the hybridomas for the production of antibodies that specifically bind to the target antibody or antigen-binding fragment thereof or chimeric antigen receptor comprising the antigen-binding fragment; and
(iii) Antibodies from hybridomas producing specifically binding antibodies are sequenced, thereby identifying anti-idiotype antibodies.
137. The method of any one of embodiments 128-136, wherein the target antibody binds to CD 19. 138. The method of any one of embodiments 128-137, wherein the antigen-binding fragment of the target antibody is derived from antibody SJ25C1, optionally wherein the antigen-binding fragment of the target antibody comprises the heavy chain variable region set forth in SEQ ID No. 23 and/or the light chain variable region set forth in SEQ ID No. 24.
139. The method of any one of embodiments 128-138, wherein the antigen-binding fragment of the target antibody is a single chain variable fragment (scFv) derived from antibody SJ25C1, optionally wherein the scFv comprises the amino acid sequence set forth in SEQ ID No. 28.
140. The method of any one of embodiments 128-137, wherein the antigen-binding fragment of the target antibody is derived from antibody FMC63, optionally wherein the antigen-binding fragment of the target antibody comprises the heavy chain variable region set forth in SEQ ID No. 30 and/or the light chain variable region set forth in SEQ ID No. 31.
141. The method of any one of embodiments 128-137 and 140, wherein the antigen-binding fragment of the target antibody is a single chain variable fragment (scFv) derived from antibody FMC63, optionally wherein the scFv comprises the amino acid sequence set forth in SEQ ID No. 34.
142. A method of depleting cells comprising administering to a subject a composition comprising an anti-idiotype antibody of any of embodiments 1-11 and 29-32, 34-37 and 39-60, or an antigen-binding fragment thereof, or a conjugate of any of embodiments 61-64, that specifically binds to a target antibody (which is antibody SJ25C1 or an antigen-binding fragment thereof), wherein the subject has been administered a cell that expresses a Chimeric Antigen Receptor (CAR) comprising the target antibody, which is antibody SJ25C1 or an antigen-binding fragment thereof.
143. A method of depleting cells comprising administering to a subject a composition comprising an anti-idiotype antibody of any of embodiments 12-31, 33-36, and 38-60, or an antigen-binding fragment thereof, or a conjugate of any of embodiments 61-64, that specifically binds to a target antibody (which is antibody FMC63 or an antigen-binding fragment thereof), wherein the subject has been administered a cell that expresses a Chimeric Antigen Receptor (CAR) comprising the target antibody, which is antibody FMC63 or an antigen-binding fragment thereof.
144. The method of embodiment 109, wherein the depleting occurs by antibody dependent cell-mediated cytotoxicity (ADCC).
145. A method of determining the presence or absence of a molecule that binds to a Chimeric Antigen Receptor (CAR), the method comprising:
(a) Contacting a binding reagent with a sample from a subject to which a cell therapy has been administered, the cell therapy comprising a cell engineered with a CAR comprising a target antibody that is antibody SJ25C1 or an antigen binding fragment thereof, under conditions that form a complex comprising the binding reagent and a molecule from the sample that binds to the binding reagent, wherein the binding reagent comprises an extracellular domain of the CAR comprising the target antibody or an antigen binding fragment thereof, or a portion thereof; and
(b) Detecting the presence or absence of the complex, thereby determining the presence or absence of a molecule that binds to the CAR.
146. The method of embodiment 145, further comprising performing steps (a) and (b) on a positive control sample, and optionally determining the presence or absence of the molecule by comparison to a positive control sample, wherein the positive control sample comprises the anti-idiotype antibody of any one of embodiments 1-11 and 29-32, 34-37 and 39-60, or an antigen-binding fragment thereof, or the conjugate of any one of embodiments 61-64, that specifically binds to a target antibody, or an antigen-binding fragment thereof.
147. A method of determining the presence or absence of a molecule that binds to a Chimeric Antigen Receptor (CAR), the method comprising:
(a) Contacting a binding reagent with a sample from a subject to which a cell therapy has been administered under conditions that form a complex comprising the binding reagent and a molecule from the sample that binds to the binding reagent, the cell therapy comprising a cell engineered with a CAR comprising a target antibody that is antibody FMC63 or an antigen binding fragment thereof, wherein the binding reagent comprises an extracellular domain or a portion of the extracellular domain of a CAR comprising the target antibody or antigen binding fragment thereof;
(b) Detecting the presence or absence of the complex.
148. The method of embodiment 147, further comprising performing steps (a) and (b) on a positive control sample, and optionally determining the presence or absence of the molecule by comparison to a positive control sample, wherein the positive control sample comprises the anti-idiotype antibody or antigen-binding fragment thereof of any of embodiments 12-31, 33-36, and 38-60 or the conjugate of any of embodiments 61-64, which specifically binds to the target antibody or antigen-binding fragment thereof.
149. The method of any of embodiments 145-148, wherein the molecule bound to the binding reagent is or comprises an antibody.
150. The method of any of embodiments 145-149, wherein the binding reagent is labeled with or capable of producing a detectable signal.
151. The method of any of embodiments 145-150, wherein the binding reagent is bound to or soluble in a solid support.
152. The method of any one of embodiments 145-153, wherein the complex is detected by an immunoassay.
153. The method of embodiment 152, wherein the immunoassay is an enzyme-linked immunosorbent assay (ELISA), chemiluminescent assay, electrochemiluminescent assay, surface Plasmon Resonance (SPR) based biosensor (e.g., BIAcore), flow cytometry, or Western blot.
154. The method of embodiment 152 or embodiment 153, wherein the immunoassay comprises mesoscale discovery.
155. The method of any of embodiments 152-154, wherein the immunoassay is a sandwich assay or a bridging assay.
156. The method of any of embodiments 145-155, wherein the binding reagent is a first binding reagent, and detecting the presence or absence of the complex comprises:
(i) Contacting the complex formed in step (a) with a second binding agent, wherein the second binding agent (1) comprises an extracellular domain of a CAR comprising a target antibody or antigen-binding fragment thereof, or a portion thereof, and (2) is detectably labeled or capable of producing a detectable signal; and
(ii) Assessing the presence or absence of the detectable signal.
157. The method of embodiment 156, wherein:
the first binding reagent is bound to a solid support, optionally wherein the first binding reagent is directly or indirectly linked to biotin and/or bound to a solid support via streptavidin; and/or
The second binding reagent is soluble.
158. The method of embodiment 156 or embodiment 157, wherein the extracellular domains of the CARs of the first and second binding agents, or portions thereof, are the same.
159. The method of any one of embodiments 150-158, wherein:
the detectable label is or comprises a fluorescent label, a chemiluminescent label, an electroluminescent label, a colorimetric label, a bioluminescent label, or a radioactive label; and/or
The detectable signal is or comprises a fluorescent signal, a chemiluminescent signal, an electroluminescent signal, a colorimetric signal, a bioluminescent signal, or a radioactive signal.
160. The method of any one of embodiments 150-159, wherein the detectable label is or comprises a SULFO tag.
161. The method of any of embodiments 145-160, wherein the antigen-binding fragment of the target antibody comprises the variable heavy chain region and/or the variable light chain region of the target antibody.
162. The method of embodiments 145-161, wherein the antigen-binding fragment of the target antibody is a single-chain fragment.
163. The method of any of embodiments 145-162, wherein the antigen-binding fragment of the target antibody is a scFv.
164. The method of any of embodiments 145-163, wherein the sample comprises whole blood, serum, or plasma.
165. An article of manufacture comprising the anti-idiotype antibody of any of embodiments 1-11 and 29-32, 34-37 and 39-60, or an antigen-binding fragment thereof, or the conjugate of any of embodiments 61-64, and instructions for using the anti-idiotype antibody for detecting a SJ25C1 antibody or antigen-binding fragment thereof, or a chimeric antigen receptor comprising a SJ25C1 antibody or antigen-binding fragment thereof; selecting or enriching from a population of cells an engineered cell expressing a Chimeric Antigen Receptor (CAR) comprising antibody SJ25C1 or an antigen binding fragment thereof; an input composition comprising cells expressing a chimeric antigen receptor comprising an SJ25C1 antibody or antigen binding fragment thereof is stimulated.
166. An article of manufacture comprising the anti-idiotype antibody of any of embodiments 12-31, 33-36, and 38-60, or an antigen-binding fragment thereof, or the conjugate of any of embodiments 61-64, and instructions for using the anti-idiotype antibody for detecting an FMC63 antibody, or an antigen-binding fragment thereof, or a chimeric antigen receptor comprising an FMC63 antibody, or an antigen-binding fragment thereof; selecting or enriching from a population of cells an engineered cell expressing a Chimeric Antigen Receptor (CAR) comprising the antibody FMC63 or an antigen binding fragment thereof; an input composition comprising cells expressing a chimeric antigen receptor comprising an FMC63 antibody or antigen binding fragment thereof is stimulated.
167. An article of manufacture, comprising:
a binding agent comprising an extracellular domain of a Chimeric Antigen Receptor (CAR) comprising a target antibody (which is antibody FMC63 or an antigen binding fragment thereof), said extracellular domain or portion thereof comprising said target antibody or antigen binding fragment thereof; and
the anti-idiotype antibody or antigen-binding fragment of any of embodiments 12-31, 33-36, and 38-60 or the conjugate of any of embodiments 61-64.
168. The article of embodiment 167, wherein the binding agent is a first binding agent, and the article further comprises a second binding agent comprising an extracellular domain of a CAR or a portion thereof.
169. The article of embodiment 167 or embodiment 168, wherein the extracellular domains of the CARs of the first and second binding agents, or portions thereof, are the same.
170. The article of any one of embodiments 167-169, further comprising instructions for using the binding reagent (optionally first and second binding reagents) to determine the presence or absence of a molecule that binds to the binding reagent in a sample by using an immunoassay, optionally wherein the immunoassay is a bridging or sandwich immunoassay, optionally wherein the sample is from a subject who has been administered a cell therapy comprising a cell engineered with a CAR comprising a target antibody that is antibody FMC63 or an antigen binding fragment thereof.
171. An article of manufacture, comprising:
a binding agent comprising an extracellular domain of a Chimeric Antigen Receptor (CAR) comprising a target antibody (which is antibody SJ25C1 or antigen binding fragment thereof), said extracellular domain or portion thereof comprising said target antibody or antigen binding fragment thereof; and
the anti-idiotype antibody or antigen-binding fragment of any of embodiments 1-11 and 29-32, 34-37 and 39-60 or the conjugate of any of embodiments 61-64.
172. The article of embodiment 171, wherein the binding agent is a first binding agent, and the article further comprises a second binding agent comprising an extracellular domain of a CAR or a portion thereof.
173. The article of embodiment 171 or embodiment 172, wherein the extracellular domains of the CARs of the first and second binding agents, or portions thereof, are the same.
174. The article of any of embodiments 171-173, further comprising instructions for using the binding reagent (optionally the first and second binding reagents) to determine the presence or absence of a molecule that binds to the binding reagent in a sample by using an immunoassay, optionally wherein the immunoassay is a bridging or sandwich immunoassay, optionally wherein the sample is from a subject who has been administered a cell therapy comprising a cell engineered with a CAR comprising a target antibody that is antibody SJ25C1 or an antigen binding fragment thereof.
175. The article of any one of embodiments 167-174, wherein the binding agent (optionally the first and/or second binding agent) is subjected to a detectable label or is capable of producing a detectable signal.
176. The article of any of embodiments 168-170 and 172-175, wherein one of the first and second binding reagents is attached to or capable of being attached to a solid support and the other of the first and second binding reagents is a detectable label or capable of producing a detectable signal.
177. The method of embodiment 176, wherein the article further comprises a solid support, optionally wherein one of the first and second binding reagents is directly or indirectly attached to biotin, and the solid support comprises a streptavidin-coated surface.
Examples
The following examples are included for illustrative purposes only and are not intended to limit the scope of the invention.
Examples1 production of anti-idiotype antibodies against SJ25C1 variable region derived antibodies
This example describes the generation of an anti-idiotype antibody (anti-ID) that recognizes the scFv portion of an exemplary anti-CD 19 Chimeric Antigen Receptor (CAR) that contains an anti-CD 19scFv having a variable heavy and a variable light chain region derived from SJ25C1 (antibody (scFv in this case) with the variable region sequences set forth in SEQ ID NOs 23 and 24 separated by the linker set forth in SEQ ID No. 25, a human CD 28-derived extracellular portion, a human CD 28-derived transmembrane domain, a human CD 28-derived intracellular signaling domain, and a human CD3 zeta-derived signaling domain.
A. Hybridoma production and antibody screening
Mice were immunized with the extracellular domain (ECD) portion of CAR (containing an anti-CD 19scFv with variable region derived from SJ25C 1). The ECD part contains a sequence
EVKLQQSGAELVRPGSSVKISCKASGYAFSSYWMNWVKQRPG QGLEWIGQIYPGDGDTNYNGKFKGQATLTADKSSSTAYMQLSGLTSEDSAVYFCARKTISSVVDFYFDYWGQGTTVTVSSGGGGSGGGGSGGGGSDIELTQSPKFMSTSVGDRVSVTCKASQNVGTNVAWYQQKPGQSPKPLIYSATYRNSGVPDRFTGSGSGTDFTLTITNVQSKDLADYFCQQYNRYPYTSGGGTKLEIKR (SEQ ID NO: 28) and extracellular portion from CD28 (SEQ ID NO: 27).
Serum isolated from immunized mice was tested by ELISA and the ability of the serum to bind to the recombinant soluble ECD moiety was tested by using secondary antibodies. Hybridoma fusion clones were generated, further characterized for binding to ECD by ELISA, and five (5) positive clones were selected. Each selected hybridoma clone was amplified and antibodies were purified. For detection of antibodies in subsequent assays, each antibody was conjugated to Alexa 647.
The ability of the antibodies to specifically bind to T cells engineered with anti-CD 19 (SJ 25C1 derived) CARs was assessed (by comparison to binding to mock transduced control T cells). T cells were isolated from human subjects by enrichment based on immunoaffinity, activated and transduced with a viral vector encoding an anti-CD 19 (SJ 25C1 derived) CAR. The surface expression of the CARs was detected by flow cytometry using a series of two-fold serial dilutions (starting at 20 μg/mL and diluted to 0.0195 μg/mL) of each anti-idiotype antibody. As a positive control, cells were also assessed for CAR surface expression using a similar concentration of goat anti-mouse ("GAM") antibody capable of detecting the murine variable region portion of the ECD portion of the CAR. No antibody controls were also tested. The dose response curve for the percentage of Alexa647 signal positive cells in T cells transduced with CAR was similar for each anti-idiotype antibody tested and comparable to the positive control GAM antibody, with none of them recognizing the mock T cells transduced with empty vector. The staining index of the anti-idiotype antibody and GAM antibody was also determined as the difference between the mean of the positive and background (simulated) peaks and the background peak spread (spin), and found to be comparable between the anti-idiotype antibody and the positive control GAM antibody. The anti-idiotype antibody clone A-1 (anti-ID A-1) was selected for further characterization.
B. Functional activity
Erk1/2 phosphorylation in Jurkat cells engineered to express the CAR described above was assessed by flow cytometry after stimulation with either an anti-ID A-1 antibody or an anti-CD 3 antibody, in each case with or without a crosslinker antibody. After incubation, the cells were fixed with formaldehyde, permeabilized, incubated with antibodies specific for phosphorylated Erk1/2, and analyzed by flow cytometry. As shown in FIG. 1, after incubation of cells in the presence of anti-ID A-1 antibodies, an increased level of Erk1/2 phosphorylation was observed similar to that observed with stimulation with anti-CD 3 antibodies in the presence of the cross-linker. Two antibodies were observed to induce a similar degree of Erk1/2 phosphorylation even in the absence of cross-linking.
Western blot was further used to compare Erk phosphorylation in CAR-expressing Jurkat cells or parent Jurkat cells that did not express CAR after stimulation with anti-ID a-1, isotype control, or anti-CD 3, or in the absence of stimulation, in the presence or absence of crosslinker antibody. The results indicate that stimulation with anti-idiotype antibodies specifically increased Erk1/2 phosphorylation in Jurkat cells transduced with anti-CD 19 (SJ 25C1 derived) CARs, but not in parental Jurkat cells (where only background signal was observed, similar to unstimulated cells and isotype control stimulated cells), to a similar extent as induced by anti-CD 3 antibodies. In contrast, anti-CD 3 antibodies induced phosphorylation in a non-specific manner (i.e., in both CAR-expressing Jurkat cells and parent Jurkat cells).
C. Sequence identification
The sequence of the anti-ID A-1 antibody was determined. Using PrimeScript TM First strand cDNA synthesis kit (Takara, catalog No. 6110A), total RNA is extracted from hybridoma cells containing hybridoma clones expressing an anti-ID A-1 antibody using an isotype-specific antisense primer or a universal primer, and cDNA is generated. RACE PCR was performed to amplify the variable (heavy and light) and constant regions of the antibodies, which were then cloned into cloning vectors and sequenced, respectively. Table 2 lists the corresponding SEQ ID NOs of the nucleotide or amino acid sequences of the antibodies.
TABLE 2 anti-ID A-1 sequences
Example 2 production of anti-idiotype antibodies against FMC 63-derived antibodies
This example describes the generation of anti-idiotype antibodies that recognize the binding domain (scFv) portion of an exemplary anti-CD 19 Chimeric Antigen Receptor (CAR) that contains an anti-CD 19scFv having VH and VL domains derived from FMC63 (an antibody containing the Variable Heavy (VH) and Variable Light (VL) sequences set forth in SEQ ID NOs 30 and 31, respectively). The scFv is set forth in SEQ ID NO. 34 and contains VH and VL regions separated by a linker set forth in SEQ ID NO. 33.
A. Hybridoma production and antibody screening
Mice were immunized with a soluble protein containing the scFv portion of the CAR (SEQ ID NO: 34) fused to the human IgG1Fc domain (SEQ ID NO:32; the protein lacks a hinge region). Soluble protein reagents for immunization are set forth in SEQ ID NO. 35.
Serum isolated from immunized mice was tested by ELISA and the ability of the serum to bind to scFv-Fc moiety was detected by using secondary antibodies. Clones were counter-screened against peptides containing only the Fc domain (SEQ ID NO: 32) to select anti-idiotype antibodies that did not cross-react with the Fc portion of scFv-Fc for immunization of mice. Clones were also counter-screened against constructs containing scfvs derived from another CD19 antibody, SJ25C1 (with variable region sequences SEQ ID nos. 23 and 24 separated by the linker set forth in SEQ ID No. 25) fused to a hingeless Fc domain (SEQ ID No. 32) to further select for anti-idiotype antibodies that do not cross-react with a different anti-CD 19 antibody.
Hybridoma fusion clones were generated and 12 candidate clones were further characterized by flow cytometry. Peripheral blood mononuclear cells are isolated from a human subject, activated and transduced with a viral vector encoding an anti-CD 19 CAR. For flow cytometry, about 1X 10 per 100. Mu.L will be used 6 Individual cells were incubated with 10 μl of biotin-conjugated anti-idiotype antibody and then stained with PE-conjugated streptavidin. As a positive control for CAR surface expression, cells were stained with anti-EGFR antibodies to verify expression of EGFRt transduction markers, which are alternatives to CAR expression. As a simulated control, PBMCs were transduced with empty vector that did not express CAR. For CD3 + CD4 + /CD8 + PBMCs gate the cells and measure fluorescent signals. The results show that the candidate anti-idiotype antibodies show specific binding to CARs on the PBMC surface. To confirm that the antibodies are specific for scFv derived from anti-CD 19 antibodies (derived from FMC 63), similar experiments were performed on cells transduced with anti-CD 19CAR derived from SJ25C1, as described in example 1. None of the candidate anti-idiotype antibodies against FMC 63-derived antibodies showed specific binding to cells expressing different anti-CD 19 CARs containing scFv derived from SJ25C 1. Anti-idiotype antibody clones B-1 (anti-ID B-1) and B-2 (anti-ID B-2) were selected for further characterization.
B. Sequence identification
The sequences of the anti-ID B-1 and B-2 antibodies were determined. Using PrimeScript TM First strand cDNA Synthesis kit (Takara, catalog number 6110A) using Isotype-specific antisense primers or universal primers, total RNA is extracted from hybridoma cells containing hybridoma clones expressing anti-ID B-1 or anti-ID B-2 antibodies, and cDNA is generated. RACE PCR was performed to amplify the variable (heavy and light) and constant regions of the antibodies, which were then cloned into cloning vectors and sequenced, respectively. Table 3 lists the corresponding SEQ ID NOs of the nucleotide or amino acid sequences of the antibodies.
TABLE 3 anti-ID B-1 and B-2 sequences
Example 3 Effect of plate-bound anti-idiotype antibodies on T cell stimulation
This example describes the results after incubation of CAR T cells in the presence of SJ25C1 specific anti-idiotype antibody (anti-ID a-1) described in example 1 or in the presence of FMC 63-derived scFv specific anti-idiotype antibody (anti-ID B-1) described in example 2.
Peripheral blood mononuclear cells are isolated from a human subject and activated and transduced with a viral vector encoding an anti-CD 19CAR having a binding domain including scFv with VH and VL domains derived from FMC63 or from SJ25C 1. Such vectors are introduced into cells to generate T cells engineered to express CARs containing FMC 63-derived scFv, and T cells engineered to express CARs containing SJ25C 1-derived scFv, respectively. In the case of FMC 63-derived CARs, the CAR coding construct further comprises a sequence encoding truncated EGFR (EGFRt) that serves as a surrogate marker for transduction and CAR expression; the EGFRt coding region is separated from the CAR coding sequence by a T2A skip sequence. The engineered cells were evaluated in various assays.
For proliferation studies, engineered T cells were labeled with 50nM carboxyfluorescein succinimidyl ester (CFSE) or CELL TRACE VIOLET (CTV) dye. In related cases, expression of the surrogate EGFRt marker was detected by staining with anti-EGFR antibody. Cells were seeded at a fixed number of cells per well in wells pre-overnight coated with 10, 5, 2.5 or 1.25 μg/ml OKT3 (anti-CD 3 antibody) or anti-idiotype antibodies recognizing SJ25C1 and FMC63 (anti-ID A-1 and anti-ID B-1, respectively) in sodium carbonate/sodium bicarbonate buffer (pH 9.0). Cells seeded in wells without antibody coating served as negative controls. Cells were cultured for 4 days and assessed for viability, proliferation and expression of CD69 and CD 25.
Proliferation of T cells expressing anti-CD 19 (SJ 25C1 or FMC 63) CARs was assessed by dye dilution using flow cytometry. CD3 observed after stimulation of T cells expressing anti-CD 19 (SJ 25C 1) CAR with anti-ID A-1 + /CFSE Low and low The percentage of cells is shown in figure 2A. CD3+/EGFR+/CFSE observed after stimulation of T cells expressing anti-CD 19 (FMC 63) CAR with anti-ID B-1 Low and low The percentage of cells is shown in figure 2B. It was observed that incubation in the presence of SJ25C 1-derived scFv specific anti-ID a-1 antibodies resulted in proliferation of T cells expressing anti-CD 19 (SJ 25C 1) CARs at levels comparable to stimulation with anti-CD 3 antibodies (fig. 2A). In contrast, proliferation observed in T cells expressing anti-CD 19 (SJ 25C 1) CARs incubated in the presence of other anti-IDs specific for different anti-CD 19CAR binding domains (FMC 63-derived scFv specific anti-Id B-1) was similar to that observed in cells without any stimulatory agent. Similarly, incubation in the presence of FMC 63-derived scFv specific anti-idiotype antibody anti-ID B-1 was observed to result in proliferation of T cells expressing anti-CD 19 (FMC 63-derived binding domain) CARs at levels comparable to stimulation with anti-CD 3 antibody. In contrast, cells incubated in the presence of another anti-ID that recognized a different anti-CD 19CAR (SJ 25C 1-derived scFv specific anti-ID a-1 antibody) were similar to those observed for cells incubated in the absence of the stimulus (fig. 2B). These results demonstrate that each of anti-ID a-1 and anti-ID B-1 is capable of stimulating and inducing proliferation of T cells expressing a CAR containing an antibody-specific corresponding scFv, but not inducing proliferation of T cells expressing a different CAR that is directed against the same antigen but does not contain the specificity of the antibody specificity Binding domain. The results are consistent with the ability of anti-ID a-1 and anti-ID B-1, i.e., anti-ID a-1 and anti-ID B-1 specifically recognize their corresponding targets without recognizing CARs containing other binding domains for the same antigen.
The results of another assay demonstrate the ability of the exemplary anti-IDs evaluated in this assay to provide CAR-specific signals to T cells in an anti-ID concentration specific manner, consistent with the utility of anti-IDs to modulate the amount of signals received via T cells expressing CD 19-specific CARs. In this assay, the mock transduced and CAR transduced T cells were labeled with CELL TRACE VIOLET (CTV). Prior to inoculation, the wells are coated with 0.25, 0.5 or 1 μg/ml of exemplary anti-idiotype antibodies specific for the binding domain of the anti-CD 19 CAR. Cells were cultured for 4 days and proliferation was assessed by assessing the extent of dye dilution via flow cytometry. Figure 2C shows anti-idiotype concentration-dependent induction of cd8+ CAR T cell proliferation. These results demonstrate the ability to modulate the amount of the plate-bound exemplary anti-idiotype antibodies provided herein in various circumstances, e.g., in order to modulate the signaling of CAR-expressing T cells and/or provide controlled levels of stimulation and/or optimize the extent of stimulation.
To further probe the stimulatory capacity of anti-ID a-1 and anti-ID B-1, flow cytometry was used to assess the expression of two T cell activation markers CD69 and CD252 following stimulation with plate-bound antibodies at concentrations of 1.25, 2.5, 5 and 10 μg/ml, transduced with target anti-CD 19CAR (SJ 25C 1-derived or FMC 63-derived, respectively). Transduced T cells were gated against EGFR as a surrogate for CAR expression and assessed for CD69 or CD25 expression by cd4+ or cd8+ egfr+ cells.
As shown in FIG. 3, for T cells transduced with anti-CD 19CAR derived from SJ25C1, anti-ID A-1 was found to be CD4 compared to cells stimulated with anti-CD 3 antibody + And CD8 + Higher CD25 expression was induced in both T cells. anti-ID A-1 also induced CD4 + And CD8 + CD69 expression in T cells was similar or slightly lower than when cells were stimulated with anti-CD 3 antibodies. CD69 and CD25 expression was not observed in unstimulated cells or cells treated with FMC 63-derived scFv specific anti-ID B-1. As shown in figure 4 of the drawings,for anti-CD 19CAR transduced T cells with FMC63, anti-ID B-1 was found to be in CD4 compared to cells stimulated with anti-CD 3 antibody + EGFR+T cells and CD8 + Higher CD25 expression was induced in both egfr+ T cells. The anti-ID B-1 antibody also induced higher levels of CD69 expression in CD4+/EGFR+ T cells than stimulated with anti-CD 3 antibody, and at similar or slightly higher levels than induced in CD8+/EGFRT+ T cells. CD69 expression in cd8+/egfr+ T cells was similar or slightly higher at higher antibody concentrations. CD69 and CD25 expression was not observed in unstimulated cells or cells treated with SJ25C1 specific anti-ID a-1. These results indicate that both anti-ID a-1 and anti-ID B-1 are capable of specifically stimulating T cells expressing CARs containing their target scFv.
EXAMPLE 4 analysis of transgene product specific host immune response
An anti-therapeutic antibody (ATA) bridging assay was developed to detect the presence or absence of antibodies recognizing the administered anti-CD 19CAR in the serum of the treated subject. Some of the anti-IDs described in examples 1-3 were used as positive controls and the ability of the assay to detect the presence of such antibodies was verified.
Biotinylated human Fc fusion protein (biotinylated ECD fusion protein) was used, which contained the scFv portion of the CAR with variable region derived from FMC63 (set forth in SEQ ID NO: 34). Adding the biotinylated ECD fusion protein to the streptavidin coated wells; plates were incubated under conditions allowing fusion proteins to bind and washed. Various concentrations of anti-ID B-1 or anti-ID B-2 were added to biotinylated fusion protein coated wells and incubated under conditions that allowed specific binding of the antibodies. After washing, ECD fusion proteins with a sulfo-tag form (sulfo-ECD fusion proteins) were produced, which contained FMC 63-derived Fc-scFv fusion. Wells were washed and Electrochemiluminescence (ECL) signals were read on a Meso Scale Discovery (MSD) Sector Imager.
As shown in FIG. 5, ECL signal increased with increasing concentrations of anti-ID B-1 and anti-ID B-2, indicating that the assay can be used to assess the presence or absence and level of antibodies in serum samples, with any of the exemplary anti-ID antibodies used as a positive control.
In some embodiments, the presence or absence of ATA antibodies to CARs in a sample from a subject that received an infusion of a dose of cell therapy containing T cells expressing an anti-CD 19 (FMC 63) CAR is assessed using an ATA assay that uses anti-ID B-1 and/or anti-ID B-2 antibodies as a positive control. In some cases, such ATA may be indicative of a host humoral immune response to the administered CAR. In an exemplary assay, plasma samples are obtained from a subject at various time points, such as before infusion and/or on days 14 and 28 after initiation of administration of a cell therapy, and in some cases, at month 3, month 6, and/or month 12. In the assays described above, samples derived from such plasma samples are used together with control samples containing anti-ID antibodies.
A similar bridging assay was also generated to evaluate samples from subjects who received an infusion of a dose of cell therapy containing T cells expressing an anti-CD 19 (SJ 25C 1) CAR. In this assay, an anti-ID A-1 antibody was used as a positive control. The assay was observed to have a sensitivity of less than 100ng/mL as determined based on the positive control anti-ID antibody and a signal 4 to 5 fold above the plasma background.
EXAMPLE 5 conjugation of anti-idiotype antibodies to beads
Either anti-ID B-1 or anti-ID-B1 as described in example 2 was covalently coupled to the surface of commercially available tosyl activated magnetic beads (ThermoFisher, waltham MA), which are superparamagnetic, nonporous, monodisperse, tosyl activated beads. The beads are covalently bound to primary amino groups and sulfhydryl groups. Conjugation was performed using beads with a diameter of about 2.8 μm (designated M-280) or 4.5 μm (designated M-450).
200 μg of anti-ID antibody was added to about 1mL of tosyl activated beads (e.g., about 4x10 with a 2.8 μm diameter) 9 Individual tosyl activated beads or about 4x10 with a 4.5 μm diameter 8 Individual tosyl activated beads) by adding to a sample containing 0.1% human serumCovalent coupling was performed by incubation in Phosphate Buffered Saline (PBS) of albumin (HSA) at 37℃overnight. The beads were washed and resuspended in 1mL PBS with 0.1% HSA. After conjugation, bead concentration was determined using a Cellometer.
To assess stability of anti-ID conjugated beads, the beads were pelleted, the supernatant removed and loaded onto a 4-12% bis-Tris SDS-PAGE gel, which was stained with coomassie blue. As a control for total protein conjugated to beads, pelleted beads were boiled in 4X (lithium dodecyl sulfate) LDS sample buffer at about 70℃for 20 minutes, and approximately 12.5. Mu.L or 25. Mu.L of boiled supernatant was also run on SDS-PAGE gels, assessed by Coomassie blue. Approximately 2.5. Mu.g or 5.0. Mu.g of anti-idiotype antibody (positive control) or 5. Mu.L of 0.1% HSA (negative control) not conjugated to the beads was also assessed by SDS-PAGE and Coomassie staining. No anti-ID antibodies were detected in the supernatant from the non-boiled conjugated samples, indicating that conjugation was stable, whereas anti-ID antibodies were detected in the supernatant from the boiled conjugated samples.
Example 6 evaluation of stimulation of T cells cultured with anti-idiotype antibody conjugated beads
FMC 63-derived scFv specific anti-ID B-1 conjugated beads were incubated with T cells. CD3 purified T cells were isolated from white blood cell apheresis samples of healthy donors by enrichment based on immunoaffinity. The isolated cells were transduced with a viral vector encoding an anti-CD 19CAR with scFv derived from FMC 63. The viral vector construct further encodes truncated EGFR (EGFRt) for use as a surrogate marker of CAR expression; the EGFRt coding region is separated from the CAR sequence by a T2A jump sequence. After transduction, the cells were expanded in a medium and frozen by cryopreservation.
For T cell stimulation studies, thawed CAR-expressing cd4+ or cd8+ cells were seeded at approximately 50,000 total cells per well, respectively. In some cases, the medium is additionally supplemented with the following cytokines: for CD4+ cells, approximately 1200IU/mL recombinant IL-7, 20IU/mL recombinant IL-15, and 100IU/mL recombinant IL-2; for CD8+ cells, approximately 200IU/mL recombinant IL-2 and 20IU/mL recombinant IL-15. anti-ID B-1 conjugated beads were added to cells at a cell to bead ratio of 1:1 or 1:5 and incubated for up to 14 days with 50% medium change every 2-3 days. As a positive control, cells were incubated with anti-CD 3/anti-CD 28 magnetic beads at a 3:1 ratio of cell to bead in the presence or absence of the indicated cytokines.
At various times during the culture, cd4+ or cd8+ transduced cells were assessed for expansion, PD-1 expression and viability (e.g., by anti-EGFR detection of expression of surrogate markers).
As shown in fig. 6 and 7, expansion of cd4+ and cd8+ T cells, particularly in the presence of cytokines, was observed when cells were cultured with anti-ID conjugated beads at a cell to bead ratio of 1:1 or 1:5, respectively. The degree of expansion was higher than when cells were cultured with control anti-CD 3/anti-CD 28 magnetic beads.
Surface expression of PD-1 on cd4+ cell subsets was assessed by flow cytometry on days 3, 7, 10 and 14 of culture. As shown in fig. 8, PD-1 expression was high on cells cultured with anti-CD 3/anti-CD 28 magnetic beads, however, PD-1 levels were significantly lower or undetectable in cells cultured with anti-ID conjugated beads.
When cells were further cultured in the presence of cytokines, cd4+ and cd8+ T cells were cultured with anti-ID conjugated beads or control anti-CD 3/anti-CD 28 conjugated beads at a 1:1 or 1:5 ratio, the percent viability of which consistently remained high during the culture period, as shown in figure 9. However, under all conditions, cell viability was reduced without the addition of cytokines, with a maximum loss of cell viability occurring in the last days of cell culture.
Example 7 assessment of cytokine production by T cells cultured with anti-idiotype antibody conjugated beads
T cells engineered with anti-CD 19CAR having scFv derived from FMC63 were generated substantially as described in example 6. Thawed CD8+ T cells were incubated with anti-ID B-1 conjugated beads in the presence of a Golgi inhibitor for 4 hours. By flow cytometry, CARs were determined + T cell subsets (e.g. generalPositive surface staining determination of alternative EGFRt transduction markers over-used against EGFR) or CAR - Intracellular cytokine levels of tnfα, ifnγ and IL-2 in T cell subsets (as determined by negative surface staining with EGFR-resistant EGFRt). As a comparison, car+ T cells (egfr+) were also cultured with CD19 expressing target cells (K562 cells transduced to express CD19, K562-CD 19) at a 1:2 effector to T cell ratio.
As shown in fig. 10A, when cells were cultured in the presence of anti-ID B-1 conjugated beads, the CAR was isolated from the cells + T cells (EGFRt) + ) Intracellular cytokine levels of TNF alpha, IFN gamma and IL-2 cytokines are induced in the CAR - T cells (EGFR) - ) And not the other. In this study, the extent of stimulation observed in the presence of anti-ID conjugated beads was similar to that observed with antigen-expressing K562-CD19 cells that were the alternative CAR-specific stimulating agent (fig. 10B) to stimulate car+t cells. These results demonstrate that anti-ID conjugated to the beads is agonistic and specifically stimulates T cells expressing CARs with antigen binding domains recognized by the anti-ID antibodies. In addition, bead reagents provide better CAR-specific stimulatory reagents than cell lines, which require cell culture and are prone to batch-to-batch variability.
Example 8 evaluation of amplification after continuous restimulation
The ability of cells to expand ex vivo after repeated stimulation may be a potential surrogate for car+ T Cell persistence (e.g., after initial activation) and/or indicative of in vivo function (Zhao et al (2015) Cancer Cell, 28:415-28). Car+ T cells were generated as described above and thawed CAR-expressing cd4+ or cd8+ T cells were plated at 50,000 car+ cells/well, respectively. anti-ID B-1 conjugated beads were added to cells at a cell to bead ratio of 1:1 or 1:5 in the presence or absence of cytokines as described in example 6. As a control, anti-CD 3/anti-CD 28 magnetic beads were added to cells at a 3:1 ratio of cell to bead with or without cytokines. Cells were harvested every 3-4 days and counted, and after resetting the cell number to the initial seeding density for each round, re-stimulated with new target cells using the same culture conditions. A total of 4 rounds of stimulation were performed during the 14 day incubation period. For each round of stimulation, the doubling number is determined.
As shown in fig. 11, sustained cell expansion of CAR-expressing (egfr+) cd4+ cells was observed after restimulation with anti-ID conjugated beads, but to a greater extent when the cells were cultured in the presence of cytokines. Also, the degree of expansion was higher than when cells were cultured with anti-CD 3/anti-CD 28 beads. For cd8+ T cells, similar kinetics of expansion were observed when the cells were cultured in the presence of anti-ID conjugated beads or anti-CD 3/anti-CD 28 beads, although the degree of expansion was slightly higher when the cells were cultured with anti-ID conjugated beads, especially in the absence of added recombinant cytokines.
Example 9 further analysis of CAR-specific cell expansion using anti-idiotype antibody conjugated beads
A similar study as described in examples 7 and 8 was performed, except that CAR-expressing T cells generated from two different patient donors were used. CD3 purified T cells were isolated from Peripheral Blood Mononuclear Cells (PBMCs) of two donor patients, transduced with viral vectors encoding anti-CD 19CAR with scFv derived from FMC63, expanded in culture, frozen, and thawed.
Thawed CD4+, CD8+ or CD4/CD8 co-cultures (1:1 ratio) were grown at approximately 5X10 6 The total cells/well were seeded into wells of a 6-well plate, wherein the medium was additionally supplemented with the following cytokines: for CD4+ cells or CD4+/CD8+ co-cultures, the medium was supplemented with approximately 1200IU/mL recombinant IL-7, 20IU/mL recombinant IL-15, and 100IU/mL recombinant IL-2; for CD8+ cells, the medium was supplemented with 200IU/mL recombinant IL-2 and 20IU/mL recombinant IL-15. anti-ID B-1 conjugated beads were added to cells at a 1:1 ratio of cells to beads and incubated for up to 9 days with 50% medium change every 2-3 days.
At various times during the culture, the number of cd4+ or cd8+ transduced cells present in the culture at each condition (by anti-EGFR detection of expression of surrogate markers) was assessed and the fold or frequency of expansion (as a percentage of total cells) of cells expressing the CAR was determined. The expression and cell viability of PD-1 and CD25 were also determined.
As shown in fig. 12A, more than 60-fold expansion of CAR-expressing (egfrt+) cd4+ T cells was observed when cd4+ cells were cultured with anti-ID conjugated beads alone. For cd8+ T cells, significantly higher expansion of CAR-expressing (egfrt+) cd8+ T cells occurred in the presence of anti-ID conjugated beads when cd8+ T cells were co-cultured with cd4+ cells. As shown in fig. 12B, the frequency of cd4+ or cd8+ expressing CAR (egfrt+) was increased during 9 days of culture in the presence of anti-ID conjugated beads, with greater than 90% of transduced cells (egfrt+) present in the culture on day 9. The viability of cd4+ and cd8+ cells also remained near 100% during the culture, with slightly higher viability observed when cd8+ T cells were co-cultured with cd4+ T cells (fig. 12C). Similar results were observed for both donors.
Surface expression of PD-1 and CD25 on transduced (egfrt+) cd4+ or cd8+ cells was assessed by flow cytometry on days 5, 7 and 9 of culture with anti-ID conjugated beads. As shown in fig. 13A, PD-1 expression on both cd4+ and cd8+ T cells decreased significantly over time with anti-ID conjugated beads. As shown in fig. 13B, CD25 expression also decreased after 9 days of incubation in the presence of anti-ID conjugated beads. Similar results were observed for both donors.
Example 10 comparison of cytokine levels and phenotypes after incubation with anti-idiotype antibody conjugated beads
CD3 purified T cells were isolated from Peripheral Blood Mononuclear Cells (PBMCs) of two donor patients, transduced with viral vectors encoding anti-CD 19 CARs with scFv derived from FMC63, and expanded by culture with beads coated with either anti-CD 3 antibody and anti-CD 28 antibody. After expansion, the expanded T cells are frozen by cryopreservation. For the study, frozen T cells were thawed and the intracellular cytokine levels or surface phenotypes of cd4+ and cd8+ T cells were assessed (d=0), or thawed cd4+, cd8+ or cd4+/cd8+ co-cultured T cells were cultured in the presence of anti-ID B-1 conjugated beads for an additional 9 days, followed by assessment of intracellular cytokine levels and surface marker phenotypes (d=9) following stimulation of K562 cells transduced with PMA/ionomycin or CD 19.
To assess intracellular cytokine levels, golgi inhibitors were added for 4 hours, and tnfα, ifnγ, and IL-2 were then assessed by flow cytometry. For all conditions, intracellular cytokine expression was significantly higher when cells were stimulated with PMA/ionomycin compared to CAR-specific stimulation of K562 cells transduced with CD 19. As shown in FIG. 14A, the levels of TNF α and IL-2 cytokines in CD4+ or CD8+ cells immediately after thawing were similar compared to the co-cultures of corresponding CD4+ or CD8+ cells or CD4/CD8T cells further cultured for 9 days in the presence of anti-ID conjugated beads. An increase in ifnγ levels was observed in cd4+, cd8+ or CD4/CD8 co-cultured T cells after thawing in the presence of anti-ID conjugated beads for a further 9 days compared to ifnγ levels in cd4+ or cd8+ T cells immediately after thawing. These results demonstrate that T cell function is maintained after 9 days of expansion with anti-ID conjugated beads. Similar results were obtained in cells from both donors.
Surface expression of activation marker CD25, inhibitory receptors PD-1 and LAG-3, and nuclear expression of proliferation marker Ki-67 in cd4+ or cd8+ cells, were also assessed immediately after thawing (d=0), or after an additional 9 days of culture in the presence of anti-ID conjugated beads (d=9) in individually expanded cd4+ or cd8+ T cells or in CD4/CD8 co-cultures. As shown in FIG. 14B, a decrease in CD25 expression was observed in cells further cultured for 9 days in the presence of anti-ID conjugated beads, as compared to T cells immediately after thawing, whereas Ki-67 was not. The reduction in CD25 expression in cd8+ cells was significantly more than in cd4+ cells. In addition, reduced expression of PD-1 and LAG-3 was also observed in both CD4+ and CD8+ cells cultured alone or in the CD4/CD8 co-culture after 9 days incubation with anti-ID conjugated beads, as compared to expression in cells immediately after thawing. This result demonstrates that previously frozen transduced cells retain functional capacity after incubation with anti-ID conjugated beads, as demonstrated by a high percentage of marker Ki-67 (indicative of cell proliferation) positive cells, but also exhibit different activation states characterized by low surface expression of CD25 activation marker and inhibitory receptor markers PD-1 and LAG-3.
Example 11 CAR-expressing cells were detected with anti-idiotype antibodies.
T cell compositions containing CAR-T cells expressing an anti-CD 19CAR containing a CD19scFv with a variable region derived from an FMC63 antibody, an immunoglobulin spacer, a transmembrane domain derived from CD28, a costimulatory region derived from 4-1BB, and a CD3 zeta intracellular signaling domain were produced. The CAR-encoding viral construct further encodes an EGFRt surrogate marker separated from the CAR sequence by a T2A jump sequence.
Cells expressing the anti-CD 19CAR were incorporated into cell samples containing healthy human Peripheral Blood Mononuclear Cells (PBMCs). The resulting cell compositions were incubated with different concentrations of anti-CD 19FMC63scFv specific anti-ID B1 or anti-ID B2 antibodies. As a control, samples of each condition were also incubated with a concentration of anti-EGFR antibodies that were able to detect EGFRt surrogate markers on transduced cells. Control samples included samples containing PBMCs alone without the addition of additional CAR-expressing T cells. Geometric Mean Fluorescence Intensity (MFI) in positively labeled cells was quantified to assess antibody staining.
As shown in fig. 15A, both anti-idiotype antibodies detected T cells with anti-CD 19CAR with scFv with variable regions derived from FMC63 in a concentration-dependent manner. As shown in fig. 15B, positive cells were detected by anti-ID B1 and anti-ID B2 antibodies in the composition containing CAR-expressing cells, whereas not in the composition containing PBMCs alone. Staining for EGFRt showed that the proportion of cells expressing the transgene to PBMCs was consistent under all conditions. The results demonstrate the ability of anti-ID B1 and anti-ID B2 antibodies to specifically detect cells expressing anti-CD 19FMC63scFv CARs, even in samples containing human PBMCs that do not express CARs. The results are consistent with the following explanation: the anti-idiotype antibody can be used to detect CAR-expressing cells in a sample from a subject (to which CAR-expressing cells that have been administered are recognized by the antibody), such as a peripheral blood sample collected from a human subject, for example, to measure the expansion, transport, and/or persistence of such cells in various tissues and/or body fluids of the subject over time.
The present invention is not intended to be limited in scope by the specific disclosed embodiments, which are provided, for example, to illustrate various aspects of the invention. Various modifications to the compositions and methods will be apparent from the description and teachings herein. Such changes may be made without departing from the true scope and spirit of the disclosure, and such changes are intended to fall within the scope of the disclosure.
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Sequence listing
<110> cinnological treatment company (Juno Therapeutics, inc.)
HAUSKINS, Colin
HEIPEL, Mark D.
SUTHERLAND, Claire L.
SATHALIYA, Taher
SMITH, Jeff
<120> anti-idiotype antibodies and related methods
<130> 735042006540
<140> not yet allocated
<141> at the same time submit
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<151> 2016-07-29
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20 25 30
Tyr Pro Lys Asp Ile Asn Val Lys Trp Lys Ile Asp Gly Ser Glu Arg
35 40 45
Gln Asn Gly Val Leu Asn Ser Trp Thr Asp Gln Asp Ser Lys Asp Ser
50 55 60
Thr Tyr Ser Met Ser Ser Thr Leu Thr Leu Thr Lys Asp Glu Tyr Glu
65 70 75 80
Arg His Asn Asn Tyr Thr Cys Glu Ala Thr His Lys Thr Ser Thr Ser
85 90 95
Pro Ile Val Lys Ser Phe Asn Arg Asn Glu Cys
100 105
<210> 7
<211> 214
<212> PRT
<213> artificial sequence
<220>
<223> anti-ID light chain
<400> 7
Asp Ile Gln Met Thr Gln Thr Thr Ser Ser Leu Ser Ala Ser Leu Gly
1 5 10 15
Asp Arg Val Thr Ile Ser Cys Arg Ala Ser Gln Asp Ile Ser Asn Tyr
20 25 30
Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr Val Lys Leu Leu Ile
35 40 45
Tyr Tyr Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile Ser Asn Leu Glu Gln
65 70 75 80
Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Gly Lys Thr Val Pro Phe
85 90 95
Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys Arg Ala Asp Ala Ala
100 105 110
Pro Thr Val Ser Ile Phe Pro Pro Ser Ser Glu Gln Leu Thr Ser Gly
115 120 125
Gly Ala Ser Val Val Cys Phe Leu Asn Asn Phe Tyr Pro Lys Asp Ile
130 135 140
Asn Val Lys Trp Lys Ile Asp Gly Ser Glu Arg Gln Asn Gly Val Leu
145 150 155 160
Asn Ser Trp Thr Asp Gln Asp Ser Lys Asp Ser Thr Tyr Ser Met Ser
165 170 175
Ser Thr Leu Thr Leu Thr Lys Asp Glu Tyr Glu Arg His Asn Asn Tyr
180 185 190
Thr Cys Glu Ala Thr His Lys Thr Ser Thr Ser Pro Ile Val Lys Ser
195 200 205
Phe Asn Arg Asn Glu Cys
210
<210> 8
<211> 20
<212> PRT
<213> artificial sequence
<220>
<223> anti-ID LC Signal sequence
<400> 8
Met Met Ser Ser Ala Gln Phe Leu Gly Leu Leu Leu Leu Cys Phe Gln
1 5 10 15
Gly Thr Arg Cys
20
<210> 9
<211> 5
<212> PRT
<213> artificial sequence
<220>
<223> anti-ID HC-CDR1
<400> 9
Ser Tyr Trp Met His
1 5
<210> 10
<211> 17
<212> PRT
<213> artificial sequence
<220>
<223> anti-ID HC-CDR2
<400> 10
Asn Ile Tyr Pro Gly Ser Gly Gly Thr Asn Tyr Asp Glu Lys Phe Lys
1 5 10 15
Arg
<210> 11
<211> 13
<212> PRT
<213> artificial sequence
<220>
<223> anti-ID HC-CDR3
<400> 11
Glu Val Thr Thr Val Ala Tyr Tyr Tyr Ser Met Asp Tyr
1 5 10
<210> 12
<211> 11
<212> PRT
<213> artificial sequence
<220>
<223> anti-ID LC-CDR1
<400> 12
Arg Ala Ser Gln Asp Ile Ser Asn Tyr Leu Asn
1 5 10
<210> 13
<211> 7
<212> PRT
<213> artificial sequence
<220>
<223> anti-ID LC-CDR2
<400> 13
Tyr Thr Ser Arg Leu His Ser
1 5
<210> 14
<211> 9
<212> PRT
<213> artificial sequence
<220>
<223> anti-ID LC-CDR3
<400> 14
Gln Gln Gly Lys Thr Val Pro Phe Thr
1 5
<210> 15
<211> 366
<212> DNA
<213> artificial sequence
<220>
<223> anti-ID VH
<400> 15
caggtccaac tgcaacaacc tgggtctgag ctggtgaggc ctggaggttc agtgaagctg 60
tcctgcaagg cttctgacta cactttcacc agctactgga tgcactgggt gaggcagagg 120
cctggacaag gccttgagtg gattggaaat atttatcctg gtagtggtgg tactaactac 180
gatgagaagt tcaagaggaa ggccacactg actgtagaca catcctccag cacagcctac 240
atgcagctcc gcagcctgac atctgaggac tctgcggtct attactgtac aagagaggtt 300
actacagtag cttattacta ttctatggac tactggggtc aaggaacctc agtcaccgtc 360
tcctca 366
<210> 16
<211> 975
<212> DNA
<213> artificial sequence
<220>
<223> anti-ID CH
<400> 16
gccaaaacga cacccccatc tgtctatcca ctggcccctg gatctgctgc ccaaactaac 60
tccatggtga ccctgggatg cctggtcaag ggctatttcc ctgagccagt gacagtgacc 120
tggaactctg gatccctgtc cagcggtgtg cacaccttcc cagctgtcct gcagtctgac 180
ctctacactc tgagcagctc agtgactgtc ccctccagca cctggcccag cgagaccgtc 240
acctgcaacg ttgcccaccc ggccagcagc accaaggtgg acaagaaaat tgtgcccagg 300
gattgtggtt gtaagccttg catatgtaca gtcccagaag tatcatctgt cttcatcttc 360
cccccaaagc ccaaggatgt gctcaccatt actctgactc ctaaggtcac gtgtgttgtg 420
gtagacatca gcaaggatga tcccgaggtc cagttcagct ggtttgtaga tgatgtggag 480
gtgcacacag ctcagacgca accccgggag gagcagttca acagcacttt ccgctcagtc 540
agtgaacttc ccatcatgca ccaggactgg ctcaatggca aggagttcaa atgcagggtc 600
aacagtgcag ctttccctgc ccccatcgag aaaaccatct ccaaaaccaa aggcagaccg 660
aaggctccac aggtgtacac cattccacct cccaaggagc agatggccaa ggataaagtc 720
agtctgacct gcatgataac agacttcttc cctgaagaca ttactgtgga gtggcagtgg 780
aatgggcagc cagcggagaa ctacaagaac actcagccca tcatggacac agatggctct 840
tacttcgtct acagcaagct caatgtgcag aagagcaact gggaggcagg aaatactttc 900
acctgctctg tgttacatga gggcctgcac aaccaccata ctgagaagag cctctcccac 960
tctcctggta aatga 975
<210> 17
<211> 1341
<212> DNA
<213> artificial sequence
<220>
<223> anti-ID heavy chain
<400> 17
caggtccaac tgcaacaacc tgggtctgag ctggtgaggc ctggaggttc agtgaagctg 60
tcctgcaagg cttctgacta cactttcacc agctactgga tgcactgggt gaggcagagg 120
cctggacaag gccttgagtg gattggaaat atttatcctg gtagtggtgg tactaactac 180
gatgagaagt tcaagaggaa ggccacactg actgtagaca catcctccag cacagcctac 240
atgcagctcc gcagcctgac atctgaggac tctgcggtct attactgtac aagagaggtt 300
actacagtag cttattacta ttctatggac tactggggtc aaggaacctc agtcaccgtc 360
tcctcagcca aaacgacacc cccatctgtc tatccactgg cccctggatc tgctgcccaa 420
actaactcca tggtgaccct gggatgcctg gtcaagggct atttccctga gccagtgaca 480
gtgacctgga actctggatc cctgtccagc ggtgtgcaca ccttcccagc tgtcctgcag 540
tctgacctct acactctgag cagctcagtg actgtcccct ccagcacctg gcccagcgag 600
accgtcacct gcaacgttgc ccacccggcc agcagcacca aggtggacaa gaaaattgtg 660
cccagggatt gtggttgtaa gccttgcata tgtacagtcc cagaagtatc atctgtcttc 720
atcttccccc caaagcccaa ggatgtgctc accattactc tgactcctaa ggtcacgtgt 780
gttgtggtag acatcagcaa ggatgatccc gaggtccagt tcagctggtt tgtagatgat 840
gtggaggtgc acacagctca gacgcaaccc cgggaggagc agttcaacag cactttccgc 900
tcagtcagtg aacttcccat catgcaccag gactggctca atggcaagga gttcaaatgc 960
agggtcaaca gtgcagcttt ccctgccccc atcgagaaaa ccatctccaa aaccaaaggc 1020
agaccgaagg ctccacaggt gtacaccatt ccacctccca aggagcagat ggccaaggat 1080
aaagtcagtc tgacctgcat gataacagac ttcttccctg aagacattac tgtggagtgg 1140
cagtggaatg ggcagccagc ggagaactac aagaacactc agcccatcat ggacacagat 1200
ggctcttact tcgtctacag caagctcaat gtgcagaaga gcaactggga ggcaggaaat 1260
actttcacct gctctgtgtt acatgagggc ctgcacaacc accatactga gaagagcctc 1320
tcccactctc ctggtaaatg a 1341
<210> 18
<211> 57
<212> DNA
<213> artificial sequence
<220>
<223> anti-ID HC Signal sequence
<400> 18
atgggatgga gctctatcat cctcttcttg gtagcaacag cctcaggtgt ccactcc 57
<210> 19
<211> 321
<212> DNA
<213> artificial sequence
<220>
<223> anti-ID VL
<400> 19
gatatccaga tgacacagac tacatcctcc ctgtctgcct ctctgggaga cagagtcacc 60
atcagttgca gggcaagtca ggacattagc aattatttaa actggtatca gcagaaacca 120
gatggaactg ttaaactcct gatctactac acatcaagat tacactcagg agtcccatca 180
aggttcagtg gcagtgggtc tggaacagat tattctctca ccattagcaa cctggagcaa 240
gaagatattg ccacttactt ttgtcagcag ggtaaaacgg ttccattcac gttcggctcg 300
gggacaaagt tggaaataaa a 321
<210> 20
<211> 324
<212> DNA
<213> artificial sequence
<220>
<223> anti-ID CL
<400> 20
cgggctgatg ctgcaccaac tgtatccatc ttcccaccat ccagtgagca gttaacatct 60
ggaggtgcct cagtcgtgtg cttcttgaac aacttctacc ccaaagacat caatgtcaag 120
tggaagattg atggcagtga acgacaaaat ggcgtcctga acagttggac tgatcaggac 180
agcaaagaca gcacctacag catgagcagc accctcacgt tgaccaagga cgagtatgaa 240
cgacataaca actatacctg tgaggccact cacaagacat caacttcacc cattgtcaag 300
agcttcaaca ggaatgagtg ttag 324
<210> 21
<211> 645
<212> DNA
<213> artificial sequence
<220>
<223> anti-ID light chain
<400> 21
gatatccaga tgacacagac tacatcctcc ctgtctgcct ctctgggaga cagagtcacc 60
atcagttgca gggcaagtca ggacattagc aattatttaa actggtatca gcagaaacca 120
gatggaactg ttaaactcct gatctactac acatcaagat tacactcagg agtcccatca 180
aggttcagtg gcagtgggtc tggaacagat tattctctca ccattagcaa cctggagcaa 240
gaagatattg ccacttactt ttgtcagcag ggtaaaacgg ttccattcac gttcggctcg 300
gggacaaagt tggaaataaa acgggctgat gctgcaccaa ctgtatccat cttcccacca 360
tccagtgagc agttaacatc tggaggtgcc tcagtcgtgt gcttcttgaa caacttctac 420
cccaaagaca tcaatgtcaa gtggaagatt gatggcagtg aacgacaaaa tggcgtcctg 480
aacagttgga ctgatcagga cagcaaagac agcacctaca gcatgagcag caccctcacg 540
ttgaccaagg acgagtatga acgacataac aactatacct gtgaggccac tcacaagaca 600
tcaacttcac ccattgtcaa gagcttcaac aggaatgagt gttag 645
<210> 22
<211> 60
<212> DNA
<213> artificial sequence
<220>
<223> anti-ID LC Signal sequence
<400> 22
atgatgtcct ctgctcagtt ccttggtctc ctgttgctct gttttcaagg taccagatgt 60
<210> 23
<211> 122
<212> PRT
<213> mice
<220>
<223> SJ25C1 VH
<400> 23
Glu Val Lys Leu Gln Gln Ser Gly Ala Glu Leu Val Arg Pro Gly Ser
1 5 10 15
Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ala Phe Ser Ser Tyr
20 25 30
Trp Met Asn Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile
35 40 45
Gly Gln Ile Tyr Pro Gly Asp Gly Asp Thr Asn Tyr Asn Gly Lys Phe
50 55 60
Lys Gly Gln Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr
65 70 75 80
Met Gln Leu Ser Gly Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys
85 90 95
Ala Arg Lys Thr Ile Ser Ser Val Val Asp Phe Tyr Phe Asp Tyr Trp
100 105 110
Gly Gln Gly Thr Thr Val Thr Val Ser Ser
115 120
<210> 24
<211> 108
<212> PRT
<213> mice
<220>
<223> SJ25C1 VL
<400> 24
Asp Ile Glu Leu Thr Gln Ser Pro Lys Phe Met Ser Thr Ser Val Gly
1 5 10 15
Asp Arg Val Ser Val Thr Cys Lys Ala Ser Gln Asn Val Gly Thr Asn
20 25 30
Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Pro Leu Ile
35 40 45
Tyr Ser Ala Thr Tyr Arg Asn Ser Gly Val Pro Asp Arg Phe Thr Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Thr Asn Val Gln Ser
65 70 75 80
Lys Asp Leu Ala Asp Tyr Phe Cys Gln Gln Tyr Asn Arg Tyr Pro Tyr
85 90 95
Thr Ser Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg
100 105
<210> 25
<211> 15
<212> PRT
<213> artificial sequence
<220>
<223> joint
<400> 25
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
1 5 10 15
<210> 26
<211> 5
<212> PRT
<213> artificial sequence
<220>
<223> 4GS linker
<400> 26
Gly Gly Gly Gly Ser
1 5
<210> 27
<211> 39
<212> PRT
<213> artificial sequence
<220>
<223> extracellular portion of CD28
<400> 27
Ile Glu Val Met Tyr Pro Pro Pro Tyr Leu Asp Asn Glu Lys Ser Asn
1 5 10 15
Gly Thr Ile Ile His Val Lys Gly Lys His Leu Cys Pro Ser Pro Leu
20 25 30
Phe Pro Gly Pro Ser Lys Pro
35
<210> 28
<211> 245
<212> PRT
<213> mice
<220>
<223> SJ25C1 scFv
<400> 28
Glu Val Lys Leu Gln Gln Ser Gly Ala Glu Leu Val Arg Pro Gly Ser
1 5 10 15
Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ala Phe Ser Ser Tyr
20 25 30
Trp Met Asn Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile
35 40 45
Gly Gln Ile Tyr Pro Gly Asp Gly Asp Thr Asn Tyr Asn Gly Lys Phe
50 55 60
Lys Gly Gln Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr
65 70 75 80
Met Gln Leu Ser Gly Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys
85 90 95
Ala Arg Lys Thr Ile Ser Ser Val Val Asp Phe Tyr Phe Asp Tyr Trp
100 105 110
Gly Gln Gly Thr Thr Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly
115 120 125
Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Glu Leu Thr Gln Ser
130 135 140
Pro Lys Phe Met Ser Thr Ser Val Gly Asp Arg Val Ser Val Thr Cys
145 150 155 160
Lys Ala Ser Gln Asn Val Gly Thr Asn Val Ala Trp Tyr Gln Gln Lys
165 170 175
Pro Gly Gln Ser Pro Lys Pro Leu Ile Tyr Ser Ala Thr Tyr Arg Asn
180 185 190
Ser Gly Val Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe
195 200 205
Thr Leu Thr Ile Thr Asn Val Gln Ser Lys Asp Leu Ala Asp Tyr Phe
210 215 220
Cys Gln Gln Tyr Asn Arg Tyr Pro Tyr Thr Ser Gly Gly Gly Thr Lys
225 230 235 240
Leu Glu Ile Lys Arg
245
<210> 29
<211> 4
<212> PRT
<213> artificial sequence
<220>
<223> 3GS linker
<400> 29
Gly Gly Gly Ser
1
<210> 30
<211> 120
<212> PRT
<213> mice
<220>
<223> FMC63 VH
<400> 30
Glu Val Lys Leu Gln Glu Ser Gly Pro Gly Leu Val Ala Pro Ser Gln
1 5 10 15
Ser Leu Ser Val Thr Cys Thr Val Ser Gly Val Ser Leu Pro Asp Tyr
20 25 30
Gly Val Ser Trp Ile Arg Gln Pro Pro Arg Lys Gly Leu Glu Trp Leu
35 40 45
Gly Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr Asn Ser Ala Leu Lys
50 55 60
Ser Arg Leu Thr Ile Ile Lys Asp Asn Ser Lys Ser Gln Val Phe Leu
65 70 75 80
Lys Met Asn Ser Leu Gln Thr Asp Asp Thr Ala Ile Tyr Tyr Cys Ala
85 90 95
Lys His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr Trp Gly Gln
100 105 110
Gly Thr Ser Val Thr Val Ser Ser
115 120
<210> 31
<211> 107
<212> PRT
<213> mice
<220>
<223> FMC63 VL
<400> 31
Asp Ile Gln Met Thr Gln Thr Thr Ser Ser Leu Ser Ala Ser Leu Gly
1 5 10 15
Asp Arg Val Thr Ile Ser Cys Arg Ala Ser Gln Asp Ile Ser Lys Tyr
20 25 30
Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr Val Lys Leu Leu Ile
35 40 45
Tyr His Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile Ser Asn Leu Glu Gln
65 70 75 80
Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Gly Asn Thr Leu Pro Tyr
85 90 95
Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Thr
100 105
<210> 32
<211> 220
<212> PRT
<213> artificial sequence
<220>
<223> hinge-lacking IgG1 Fc
<400> 32
Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe
1 5 10 15
Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val
20 25 30
Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe
35 40 45
Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro
50 55 60
Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr
65 70 75 80
Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val
85 90 95
Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala
100 105 110
Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg
115 120 125
Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly
130 135 140
Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro
145 150 155 160
Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser
165 170 175
Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln
180 185 190
Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His
195 200 205
Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
210 215 220
<210> 33
<211> 18
<212> PRT
<213> artificial sequence
<220>
<223> joint
<400> 33
Gly Ser Thr Ser Gly Ser Gly Lys Pro Gly Ser Gly Glu Gly Ser Thr
1 5 10 15
Lys Gly
<210> 34
<211> 245
<212> PRT
<213> artificial sequence
<220>
<223> FMC63 scFv
<400> 34
Asp Ile Gln Met Thr Gln Thr Thr Ser Ser Leu Ser Ala Ser Leu Gly
1 5 10 15
Asp Arg Val Thr Ile Ser Cys Arg Ala Ser Gln Asp Ile Ser Lys Tyr
20 25 30
Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr Val Lys Leu Leu Ile
35 40 45
Tyr His Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile Ser Asn Leu Glu Gln
65 70 75 80
Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Gly Asn Thr Leu Pro Tyr
85 90 95
Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Thr Gly Ser Thr Ser Gly
100 105 110
Ser Gly Lys Pro Gly Ser Gly Glu Gly Ser Thr Lys Gly Glu Val Lys
115 120 125
Leu Gln Glu Ser Gly Pro Gly Leu Val Ala Pro Ser Gln Ser Leu Ser
130 135 140
Val Thr Cys Thr Val Ser Gly Val Ser Leu Pro Asp Tyr Gly Val Ser
145 150 155 160
Trp Ile Arg Gln Pro Pro Arg Lys Gly Leu Glu Trp Leu Gly Val Ile
165 170 175
Trp Gly Ser Glu Thr Thr Tyr Tyr Asn Ser Ala Leu Lys Ser Arg Leu
180 185 190
Thr Ile Ile Lys Asp Asn Ser Lys Ser Gln Val Phe Leu Lys Met Asn
195 200 205
Ser Leu Gln Thr Asp Asp Thr Ala Ile Tyr Tyr Cys Ala Lys His Tyr
210 215 220
Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr Ser
225 230 235 240
Val Thr Val Ser Ser
245
<210> 35
<211> 465
<212> PRT
<213> artificial sequence
<220>
<223> FMC63 reagent
<400> 35
Asp Ile Gln Met Thr Gln Thr Thr Ser Ser Leu Ser Ala Ser Leu Gly
1 5 10 15
Asp Arg Val Thr Ile Ser Cys Arg Ala Ser Gln Asp Ile Ser Lys Tyr
20 25 30
Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr Val Lys Leu Leu Ile
35 40 45
Tyr His Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile Ser Asn Leu Glu Gln
65 70 75 80
Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Gly Asn Thr Leu Pro Tyr
85 90 95
Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Thr Gly Ser Thr Ser Gly
100 105 110
Ser Gly Lys Pro Gly Ser Gly Glu Gly Ser Thr Lys Gly Glu Val Lys
115 120 125
Leu Gln Glu Ser Gly Pro Gly Leu Val Ala Pro Ser Gln Ser Leu Ser
130 135 140
Val Thr Cys Thr Val Ser Gly Val Ser Leu Pro Asp Tyr Gly Val Ser
145 150 155 160
Trp Ile Arg Gln Pro Pro Arg Lys Gly Leu Glu Trp Leu Gly Val Ile
165 170 175
Trp Gly Ser Glu Thr Thr Tyr Tyr Asn Ser Ala Leu Lys Ser Arg Leu
180 185 190
Thr Ile Ile Lys Asp Asn Ser Lys Ser Gln Val Phe Leu Lys Met Asn
195 200 205
Ser Leu Gln Thr Asp Asp Thr Ala Ile Tyr Tyr Cys Ala Lys His Tyr
210 215 220
Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr Ser
225 230 235 240
Val Thr Val Ser Ser Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro
245 250 255
Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser
260 265 270
Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp
275 280 285
Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn
290 295 300
Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val
305 310 315 320
Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu
325 330 335
Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys
340 345 350
Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr
355 360 365
Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr
370 375 380
Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu
385 390 395 400
Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu
405 410 415
Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys
420 425 430
Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu
435 440 445
Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly
450 455 460
Lys
465
<210> 36
<211> 122
<212> PRT
<213> artificial sequence
<220>
<223> anti-ID B-1 VH
<400> 36
Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr
20 25 30
Tyr Met Lys Trp Val Lys Gln Cys His Gly Lys Ser Leu Glu Trp Ile
35 40 45
Gly Asp Ile Asn Pro Asn Asn Gly Gly Thr Asp Tyr Asn Gln Asn Phe
50 55 60
Lys Gly Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr
65 70 75 80
Met Gln Leu Asn Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Glu Gly Asn Asn Tyr Gly Ser Arg Asp Ala Met Asp Tyr Trp
100 105 110
Gly Gln Gly Thr Ser Val Thr Val Ser Ser
115 120
<210> 37
<211> 330
<212> PRT
<213> artificial sequence
<220>
<223> anti-ID B-1 CH
<400> 37
Ala Lys Thr Thr Ala Pro Ser Val Tyr Pro Leu Ala Pro Val Cys Gly
1 5 10 15
Asp Thr Thr Gly Ser Ser Val Thr Leu Gly Cys Leu Val Lys Gly Tyr
20 25 30
Phe Pro Glu Pro Val Thr Leu Thr Trp Asn Ser Gly Ser Leu Ser Ser
35 40 45
Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Asp Leu Tyr Thr Leu
50 55 60
Ser Ser Ser Val Thr Val Thr Ser Ser Thr Trp Pro Ser Gln Ser Ile
65 70 75 80
Thr Cys Asn Val Ala His Pro Ala Ser Ser Thr Lys Val Asp Lys Lys
85 90 95
Ile Glu Pro Arg Gly Pro Thr Ile Lys Pro Cys Pro Pro Cys Lys Cys
100 105 110
Pro Ala Pro Asn Leu Leu Gly Gly Pro Ser Val Phe Ile Phe Pro Pro
115 120 125
Lys Ile Lys Asp Val Leu Met Ile Ser Leu Ser Pro Ile Val Thr Cys
130 135 140
Val Val Val Asp Val Ser Glu Asp Asp Pro Asp Val Gln Ile Ser Trp
145 150 155 160
Phe Val Asn Asn Val Glu Val His Thr Ala Gln Thr Gln Thr His Arg
165 170 175
Glu Asp Tyr Asn Ser Thr Leu Arg Val Val Ser Ala Leu Pro Ile Gln
180 185 190
His Gln Asp Trp Met Ser Gly Lys Glu Phe Lys Cys Lys Val Asn Asn
195 200 205
Lys Asp Leu Pro Ala Pro Ile Glu Arg Thr Ile Ser Lys Pro Lys Gly
210 215 220
Ser Val Arg Ala Pro Gln Val Tyr Val Leu Pro Pro Pro Glu Glu Glu
225 230 235 240
Met Thr Lys Lys Gln Val Thr Leu Thr Cys Met Val Thr Asp Phe Met
245 250 255
Pro Glu Asp Ile Tyr Val Glu Trp Thr Asn Asn Gly Lys Thr Glu Leu
260 265 270
Asn Tyr Lys Asn Thr Glu Pro Val Leu Asp Ser Asp Gly Ser Tyr Phe
275 280 285
Met Tyr Ser Lys Leu Arg Val Glu Lys Lys Asn Trp Val Glu Arg Asn
290 295 300
Ser Tyr Ser Cys Ser Val Val His Glu Gly Leu His Asn His His Thr
305 310 315 320
Thr Lys Ser Phe Ser Arg Thr Pro Gly Lys
325 330
<210> 38
<211> 452
<212> PRT
<213> artificial sequence
<220>
<223> anti-ID B-1 heavy chain
<400> 38
Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr
20 25 30
Tyr Met Lys Trp Val Lys Gln Cys His Gly Lys Ser Leu Glu Trp Ile
35 40 45
Gly Asp Ile Asn Pro Asn Asn Gly Gly Thr Asp Tyr Asn Gln Asn Phe
50 55 60
Lys Gly Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr
65 70 75 80
Met Gln Leu Asn Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Glu Gly Asn Asn Tyr Gly Ser Arg Asp Ala Met Asp Tyr Trp
100 105 110
Gly Gln Gly Thr Ser Val Thr Val Ser Ser Ala Lys Thr Thr Ala Pro
115 120 125
Ser Val Tyr Pro Leu Ala Pro Val Cys Gly Asp Thr Thr Gly Ser Ser
130 135 140
Val Thr Leu Gly Cys Leu Val Lys Gly Tyr Phe Pro Glu Pro Val Thr
145 150 155 160
Leu Thr Trp Asn Ser Gly Ser Leu Ser Ser Gly Val His Thr Phe Pro
165 170 175
Ala Val Leu Gln Ser Asp Leu Tyr Thr Leu Ser Ser Ser Val Thr Val
180 185 190
Thr Ser Ser Thr Trp Pro Ser Gln Ser Ile Thr Cys Asn Val Ala His
195 200 205
Pro Ala Ser Ser Thr Lys Val Asp Lys Lys Ile Glu Pro Arg Gly Pro
210 215 220
Thr Ile Lys Pro Cys Pro Pro Cys Lys Cys Pro Ala Pro Asn Leu Leu
225 230 235 240
Gly Gly Pro Ser Val Phe Ile Phe Pro Pro Lys Ile Lys Asp Val Leu
245 250 255
Met Ile Ser Leu Ser Pro Ile Val Thr Cys Val Val Val Asp Val Ser
260 265 270
Glu Asp Asp Pro Asp Val Gln Ile Ser Trp Phe Val Asn Asn Val Glu
275 280 285
Val His Thr Ala Gln Thr Gln Thr His Arg Glu Asp Tyr Asn Ser Thr
290 295 300
Leu Arg Val Val Ser Ala Leu Pro Ile Gln His Gln Asp Trp Met Ser
305 310 315 320
Gly Lys Glu Phe Lys Cys Lys Val Asn Asn Lys Asp Leu Pro Ala Pro
325 330 335
Ile Glu Arg Thr Ile Ser Lys Pro Lys Gly Ser Val Arg Ala Pro Gln
340 345 350
Val Tyr Val Leu Pro Pro Pro Glu Glu Glu Met Thr Lys Lys Gln Val
355 360 365
Thr Leu Thr Cys Met Val Thr Asp Phe Met Pro Glu Asp Ile Tyr Val
370 375 380
Glu Trp Thr Asn Asn Gly Lys Thr Glu Leu Asn Tyr Lys Asn Thr Glu
385 390 395 400
Pro Val Leu Asp Ser Asp Gly Ser Tyr Phe Met Tyr Ser Lys Leu Arg
405 410 415
Val Glu Lys Lys Asn Trp Val Glu Arg Asn Ser Tyr Ser Cys Ser Val
420 425 430
Val His Glu Gly Leu His Asn His His Thr Thr Lys Ser Phe Ser Arg
435 440 445
Thr Pro Gly Lys
450
<210> 39
<211> 19
<212> PRT
<213> artificial sequence
<220>
<223> anti-ID B-1 HC Signal sequence
<400> 39
Met Gly Trp Ser Trp Ile Phe Leu Phe Leu Leu Ser Gly Thr Ala Gly
1 5 10 15
Val Leu Ser
<210> 40
<211> 106
<212> PRT
<213> artificial sequence
<220>
<223> anti-ID B-1 VL
<400> 40
Gln Ile Val Leu Thr Gln Ser Pro Ala Leu Met Ser Ala Ser Pro Gly
1 5 10 15
Glu Lys Val Thr Met Thr Cys Ser Ala Ser Ser Gly Val Ile Tyr Met
20 25 30
Tyr Trp Tyr Gln Gln Lys Pro Arg Ser Ser Pro Lys Pro Trp Ile Tyr
35 40 45
Leu Thr Ser Asn Leu Ala Ser Gly Val Pro Ala Arg Phe Ser Gly Ser
50 55 60
Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Ser Met Glu Ala Glu
65 70 75 80
Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Ser Asn Pro Leu Thr
85 90 95
Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys
100 105
<210> 41
<211> 107
<212> PRT
<213> artificial sequence
<220>
<223> anti-ID B-1 CL
<400> 41
Arg Ala Asp Ala Ala Pro Thr Val Ser Ile Phe Pro Pro Ser Ser Glu
1 5 10 15
Gln Leu Thr Ser Gly Gly Ala Ser Val Val Cys Phe Leu Asn Asn Phe
20 25 30
Tyr Pro Lys Asp Ile Asn Val Lys Trp Lys Ile Asp Gly Ser Glu Arg
35 40 45
Gln Asn Gly Val Leu Asn Ser Trp Thr Asp Gln Asp Ser Lys Asp Ser
50 55 60
Thr Tyr Ser Met Ser Ser Thr Leu Thr Leu Thr Lys Asp Glu Tyr Glu
65 70 75 80
Arg His Asn Ser Tyr Thr Cys Glu Ala Thr His Lys Thr Ser Thr Ser
85 90 95
Pro Ile Val Lys Ser Phe Asn Arg Asn Glu Cys
100 105
<210> 42
<211> 213
<212> PRT
<213> artificial sequence
<220>
<223> anti-ID B-1 light chain
<400> 42
Gln Ile Val Leu Thr Gln Ser Pro Ala Leu Met Ser Ala Ser Pro Gly
1 5 10 15
Glu Lys Val Thr Met Thr Cys Ser Ala Ser Ser Gly Val Ile Tyr Met
20 25 30
Tyr Trp Tyr Gln Gln Lys Pro Arg Ser Ser Pro Lys Pro Trp Ile Tyr
35 40 45
Leu Thr Ser Asn Leu Ala Ser Gly Val Pro Ala Arg Phe Ser Gly Ser
50 55 60
Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Ser Met Glu Ala Glu
65 70 75 80
Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Ser Asn Pro Leu Thr
85 90 95
Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys Arg Ala Asp Ala Ala Pro
100 105 110
Thr Val Ser Ile Phe Pro Pro Ser Ser Glu Gln Leu Thr Ser Gly Gly
115 120 125
Ala Ser Val Val Cys Phe Leu Asn Asn Phe Tyr Pro Lys Asp Ile Asn
130 135 140
Val Lys Trp Lys Ile Asp Gly Ser Glu Arg Gln Asn Gly Val Leu Asn
145 150 155 160
Ser Trp Thr Asp Gln Asp Ser Lys Asp Ser Thr Tyr Ser Met Ser Ser
165 170 175
Thr Leu Thr Leu Thr Lys Asp Glu Tyr Glu Arg His Asn Ser Tyr Thr
180 185 190
Cys Glu Ala Thr His Lys Thr Ser Thr Ser Pro Ile Val Lys Ser Phe
195 200 205
Asn Arg Asn Glu Cys
210
<210> 43
<211> 22
<212> PRT
<213> artificial sequence
<220>
<223> anti-ID B-1 LC Signal sequence
<400> 43
Met Asp Phe Gln Val Gln Ile Phe Ser Phe Leu Leu Met Ser Ala Ser
1 5 10 15
Val Ile Met Ser Arg Gly
20
<210> 44
<211> 5
<212> PRT
<213> artificial sequence
<220>
<223> anti-ID B-1 HC-CDR1
<400> 44
Asp Tyr Tyr Met Lys
1 5
<210> 45
<211> 17
<212> PRT
<213> artificial sequence
<220>
<223> anti-ID B-1 HC-CDR2
<400> 45
Asp Ile Asn Pro Asn Asn Gly Gly Thr Asp Tyr Asn Gln Asn Phe Lys
1 5 10 15
Gly
<210> 46
<211> 13
<212> PRT
<213> artificial sequence
<220>
<223> anti-ID B-1 HC-CDR3
<400> 46
Glu Gly Asn Asn Tyr Gly Ser Arg Asp Ala Met Asp Tyr
1 5 10
<210> 47
<211> 10
<212> PRT
<213> artificial sequence
<220>
<223> anti-ID B-1 LC-CDR1
<400> 47
Ser Ala Ser Ser Gly Val Ile Tyr Met Tyr
1 5 10
<210> 48
<211> 7
<212> PRT
<213> artificial sequence
<220>
<223> anti-ID B-1 LC-CDR2
<400> 48
Leu Thr Ser Asn Leu Ala Ser
1 5
<210> 49
<211> 9
<212> PRT
<213> artificial sequence
<220>
<223> anti-ID B-1 LC-CDR3
<400> 49
Gln Gln Trp Ser Ser Asn Pro Leu Thr
1 5
<210> 50
<211> 366
<212> DNA
<213> artificial sequence
<220>
<223> anti-ID B-1 VH
<400> 50
gaggtccagc tgcaacaatc tggacctgag ctggtgaagc ctggggcttc agtgaagatg 60
tcctgtaagg cttctggata cacattcact gactactaca tgaagtgggt gaagcagtgt 120
catggaaaga gccttgagtg gattggagat attaatccta acaatggtgg tactgactac 180
aaccagaact ttaagggcaa ggccacattg actgtagaca aatcctccag cacagcctac 240
atgcagctca acagcctgac atctgaggac tctgcagtct attactgtgc aagagagggg 300
aataactacg gtagtagaga tgctatggac tactggggtc aaggaacgtc agtcaccgtc 360
tcctca 366
<210> 51
<211> 990
<212> DNA
<213> artificial sequence
<220>
<223> anti-ID B-1 CH
<400> 51
gccaaaacaa cagccccatc ggtctatcca ctggcccctg tgtgtggaga tacaactggc 60
tcctcggtga ctctaggatg cctggtcaag ggttatttcc ctgagccagt gaccttgacc 120
tggaactctg gatccctgtc cagtggtgtg cacaccttcc cagctgtcct gcagtctgac 180
ctctacaccc tcagcagctc agtgactgta acctcgagca cctggcccag ccagtccatc 240
acctgcaatg tggcccaccc ggcaagcagc accaaggtgg acaagaaaat tgagcccaga 300
gggcccacaa tcaagccctg tcctccatgc aaatgcccag cacctaacct cttgggtgga 360
ccatccgtct tcatcttccc tccaaagatc aaggatgtac tcatgatctc cctgagcccc 420
atagtcacat gtgtggtggt ggatgtgagc gaggatgacc cagatgtcca gatcagctgg 480
tttgtgaaca acgtggaagt acacacagct cagacacaaa cccatagaga ggattacaac 540
agtactctcc gggtggtcag tgccctcccc atccagcacc aggactggat gagtggcaag 600
gagttcaaat gcaaggtcaa caacaaagac ctcccagcgc ccatcgagag aaccatctca 660
aaacccaaag ggtcagtaag agctccacag gtatatgtct tgcctccacc agaagaagag 720
atgactaaga aacaggtcac tctgacctgc atggtcacag acttcatgcc tgaagacatt 780
tacgtggagt ggaccaacaa cgggaaaaca gagctaaact acaagaacac tgaaccagtc 840
ctggactctg atggttctta cttcatgtac agcaagctga gagtggaaaa gaagaactgg 900
gtggaaagaa atagctactc ctgttcagtg gtccacgagg gtctgcacaa tcaccacacg 960
actaagagct tctcccggac tccgggtaaa 990
<210> 52
<211> 1356
<212> DNA
<213> artificial sequence
<220>
<223> anti-ID B-1 heavy chain
<400> 52
gaggtccagc tgcaacaatc tggacctgag ctggtgaagc ctggggcttc agtgaagatg 60
tcctgtaagg cttctggata cacattcact gactactaca tgaagtgggt gaagcagtgt 120
catggaaaga gccttgagtg gattggagat attaatccta acaatggtgg tactgactac 180
aaccagaact ttaagggcaa ggccacattg actgtagaca aatcctccag cacagcctac 240
atgcagctca acagcctgac atctgaggac tctgcagtct attactgtgc aagagagggg 300
aataactacg gtagtagaga tgctatggac tactggggtc aaggaacgtc agtcaccgtc 360
tcctcagcca aaacaacagc cccatcggtc tatccactgg cccctgtgtg tggagataca 420
actggctcct cggtgactct aggatgcctg gtcaagggtt atttccctga gccagtgacc 480
ttgacctgga actctggatc cctgtccagt ggtgtgcaca ccttcccagc tgtcctgcag 540
tctgacctct acaccctcag cagctcagtg actgtaacct cgagcacctg gcccagccag 600
tccatcacct gcaatgtggc ccacccggca agcagcacca aggtggacaa gaaaattgag 660
cccagagggc ccacaatcaa gccctgtcct ccatgcaaat gcccagcacc taacctcttg 720
ggtggaccat ccgtcttcat cttccctcca aagatcaagg atgtactcat gatctccctg 780
agccccatag tcacatgtgt ggtggtggat gtgagcgagg atgacccaga tgtccagatc 840
agctggtttg tgaacaacgt ggaagtacac acagctcaga cacaaaccca tagagaggat 900
tacaacagta ctctccgggt ggtcagtgcc ctccccatcc agcaccagga ctggatgagt 960
ggcaaggagt tcaaatgcaa ggtcaacaac aaagacctcc cagcgcccat cgagagaacc 1020
atctcaaaac ccaaagggtc agtaagagct ccacaggtat atgtcttgcc tccaccagaa 1080
gaagagatga ctaagaaaca ggtcactctg acctgcatgg tcacagactt catgcctgaa 1140
gacatttacg tggagtggac caacaacggg aaaacagagc taaactacaa gaacactgaa 1200
ccagtcctgg actctgatgg ttcttacttc atgtacagca agctgagagt ggaaaagaag 1260
aactgggtgg aaagaaatag ctactcctgt tcagtggtcc acgagggtct gcacaatcac 1320
cacacgacta agagcttctc ccggactccg ggtaaa 1356
<210> 53
<211> 57
<212> DNA
<213> artificial sequence
<220>
<223> anti-ID B-1 HC Signal sequence
<400> 53
atgggatgga gctggatctt tctcttcctc ttgtcaggaa ctgcaggtgt cctctct 57
<210> 54
<211> 318
<212> DNA
<213> artificial sequence
<220>
<223> anti-ID B-1 VL
<400> 54
caaattgttc tcacccagtc tccagcactc atgtctgcat ctccagggga gaaggtcacc 60
atgacctgca gtgccagctc aggtgtaatt tacatgtact ggtaccaaca gaagccaaga 120
tcctccccca aaccctggat ttatctcaca tccaacctgg cttctggagt ccctgctcgc 180
ttcagtggca gtgggtctgg gacctcttac tctctcacaa tcagcagcat ggaggctgaa 240
gatgctgcca cttattactg ccagcagtgg agtagtaacc cgctcacgtt cggtgctggc 300
accaagctgg agctgaaa 318
<210> 55
<211> 321
<212> DNA
<213> artificial sequence
<220>
<223> anti-ID B-1 CL
<400> 55
cgggctgatg ctgcaccaac tgtatccatc ttcccaccat ccagtgagca gttaacatct 60
ggaggtgcct cagtcgtgtg cttcttgaac aacttctacc ccaaagacat caatgtcaag 120
tggaagattg atggcagtga acgacaaaat ggcgtcctga acagttggac tgatcaggac 180
agcaaagaca gcacctacag catgagcagc accctcacgt tgaccaagga cgagtatgaa 240
cgacataaca gctatacctg tgaggccact cacaagacat caacttcacc cattgtcaag 300
agcttcaaca ggaatgagtg t 321
<210> 56
<211> 639
<212> DNA
<213> artificial sequence
<220>
<223> anti-ID B-1 light chain
<400> 56
caaattgttc tcacccagtc tccagcactc atgtctgcat ctccagggga gaaggtcacc 60
atgacctgca gtgccagctc aggtgtaatt tacatgtact ggtaccaaca gaagccaaga 120
tcctccccca aaccctggat ttatctcaca tccaacctgg cttctggagt ccctgctcgc 180
ttcagtggca gtgggtctgg gacctcttac tctctcacaa tcagcagcat ggaggctgaa 240
gatgctgcca cttattactg ccagcagtgg agtagtaacc cgctcacgtt cggtgctggc 300
accaagctgg agctgaaacg ggctgatgct gcaccaactg tatccatctt cccaccatcc 360
agtgagcagt taacatctgg aggtgcctca gtcgtgtgct tcttgaacaa cttctacccc 420
aaagacatca atgtcaagtg gaagattgat ggcagtgaac gacaaaatgg cgtcctgaac 480
agttggactg atcaggacag caaagacagc acctacagca tgagcagcac cctcacgttg 540
accaaggacg agtatgaacg acataacagc tatacctgtg aggccactca caagacatca 600
acttcaccca ttgtcaagag cttcaacagg aatgagtgt 639
<210> 57
<211> 66
<212> DNA
<213> artificial sequence
<220>
<223> anti-ID B-1 LC Signal sequence
<400> 57
atggattttc aagtgcagat tttcagcttc ctgctaatga gtgcctcagt cataatgtcc 60
agggga 66
<210> 58
<211> 115
<212> PRT
<213> artificial sequence
<220>
<223> anti-ID B-2 VH
<400> 58
Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Leu Val Arg Pro Gly Ala
1 5 10 15
Ser Val Lys Leu Ser Cys Lys Thr Ser Gly Tyr Ser Phe Thr Arg Tyr
20 25 30
Trp Met Asn Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile
35 40 45
Gly Met Ile His Pro Ser Asp Ser Glu Thr Arg Leu Asn Gln Lys Phe
50 55 60
Lys Asp Lys Ala Thr Leu Thr Val Asp Asn Ser Ser Ser Thr Ala Tyr
65 70 75 80
Met Gln Leu Ser Ser Pro Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys
85 90 95
Ala Ser Ile Tyr Tyr Glu Glu Ala Trp Gly Gln Gly Thr Leu Val Thr
100 105 110
Val Ser Ala
115
<210> 59
<211> 324
<212> PRT
<213> artificial sequence
<220>
<223> anti-ID B-2 CH
<400> 59
Ala Lys Thr Thr Pro Pro Ser Val Tyr Pro Leu Ala Pro Gly Ser Ala
1 5 10 15
Ala Gln Thr Asn Ser Met Val Thr Leu Gly Cys Leu Val Lys Gly Tyr
20 25 30
Phe Pro Glu Pro Val Thr Val Thr Trp Asn Ser Gly Ser Leu Ser Ser
35 40 45
Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Asp Leu Tyr Thr Leu
50 55 60
Ser Ser Ser Val Thr Val Pro Ser Ser Thr Trp Pro Ser Glu Thr Val
65 70 75 80
Thr Cys Asn Val Ala His Pro Ala Ser Ser Thr Lys Val Asp Lys Lys
85 90 95
Ile Val Pro Arg Asp Cys Gly Cys Lys Pro Cys Ile Cys Thr Val Pro
100 105 110
Glu Val Ser Ser Val Phe Ile Phe Pro Pro Lys Pro Lys Asp Val Leu
115 120 125
Thr Ile Thr Leu Thr Pro Lys Val Thr Cys Val Val Val Asp Ile Ser
130 135 140
Lys Asp Asp Pro Glu Val Gln Phe Ser Trp Phe Val Asp Asp Val Glu
145 150 155 160
Val His Thr Ala Gln Thr Gln Pro Arg Glu Glu Gln Phe Asn Ser Thr
165 170 175
Phe Arg Ser Val Ser Glu Leu Pro Ile Met His Gln Asp Trp Leu Asn
180 185 190
Gly Lys Glu Phe Lys Cys Arg Val Asn Ser Ala Ala Phe Pro Ala Pro
195 200 205
Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Arg Pro Lys Ala Pro Gln
210 215 220
Val Tyr Thr Ile Pro Pro Pro Lys Glu Gln Met Ala Lys Asp Lys Val
225 230 235 240
Ser Leu Thr Cys Met Ile Thr Asp Phe Phe Pro Glu Asp Ile Thr Val
245 250 255
Glu Trp Gln Trp Asn Gly Gln Pro Ala Glu Asn Tyr Lys Asn Thr Gln
260 265 270
Pro Ile Met Asp Thr Asp Gly Ser Tyr Phe Val Tyr Ser Lys Leu Asn
275 280 285
Val Gln Lys Ser Asn Trp Glu Ala Gly Asn Thr Phe Thr Cys Ser Val
290 295 300
Leu His Glu Gly Leu His Asn His His Thr Glu Lys Ser Leu Ser His
305 310 315 320
Ser Pro Gly Lys
<210> 60
<211> 439
<212> PRT
<213> artificial sequence
<220>
<223> anti-ID B-2 heavy chain
<400> 60
Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Leu Val Arg Pro Gly Ala
1 5 10 15
Ser Val Lys Leu Ser Cys Lys Thr Ser Gly Tyr Ser Phe Thr Arg Tyr
20 25 30
Trp Met Asn Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile
35 40 45
Gly Met Ile His Pro Ser Asp Ser Glu Thr Arg Leu Asn Gln Lys Phe
50 55 60
Lys Asp Lys Ala Thr Leu Thr Val Asp Asn Ser Ser Ser Thr Ala Tyr
65 70 75 80
Met Gln Leu Ser Ser Pro Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys
85 90 95
Ala Ser Ile Tyr Tyr Glu Glu Ala Trp Gly Gln Gly Thr Leu Val Thr
100 105 110
Val Ser Ala Ala Lys Thr Thr Pro Pro Ser Val Tyr Pro Leu Ala Pro
115 120 125
Gly Ser Ala Ala Gln Thr Asn Ser Met Val Thr Leu Gly Cys Leu Val
130 135 140
Lys Gly Tyr Phe Pro Glu Pro Val Thr Val Thr Trp Asn Ser Gly Ser
145 150 155 160
Leu Ser Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Asp Leu
165 170 175
Tyr Thr Leu Ser Ser Ser Val Thr Val Pro Ser Ser Thr Trp Pro Ser
180 185 190
Glu Thr Val Thr Cys Asn Val Ala His Pro Ala Ser Ser Thr Lys Val
195 200 205
Asp Lys Lys Ile Val Pro Arg Asp Cys Gly Cys Lys Pro Cys Ile Cys
210 215 220
Thr Val Pro Glu Val Ser Ser Val Phe Ile Phe Pro Pro Lys Pro Lys
225 230 235 240
Asp Val Leu Thr Ile Thr Leu Thr Pro Lys Val Thr Cys Val Val Val
245 250 255
Asp Ile Ser Lys Asp Asp Pro Glu Val Gln Phe Ser Trp Phe Val Asp
260 265 270
Asp Val Glu Val His Thr Ala Gln Thr Gln Pro Arg Glu Glu Gln Phe
275 280 285
Asn Ser Thr Phe Arg Ser Val Ser Glu Leu Pro Ile Met His Gln Asp
290 295 300
Trp Leu Asn Gly Lys Glu Phe Lys Cys Arg Val Asn Ser Ala Ala Phe
305 310 315 320
Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Arg Pro Lys
325 330 335
Ala Pro Gln Val Tyr Thr Ile Pro Pro Pro Lys Glu Gln Met Ala Lys
340 345 350
Asp Lys Val Ser Leu Thr Cys Met Ile Thr Asp Phe Phe Pro Glu Asp
355 360 365
Ile Thr Val Glu Trp Gln Trp Asn Gly Gln Pro Ala Glu Asn Tyr Lys
370 375 380
Asn Thr Gln Pro Ile Met Asp Thr Asp Gly Ser Tyr Phe Val Tyr Ser
385 390 395 400
Lys Leu Asn Val Gln Lys Ser Asn Trp Glu Ala Gly Asn Thr Phe Thr
405 410 415
Cys Ser Val Leu His Glu Gly Leu His Asn His His Thr Glu Lys Ser
420 425 430
Leu Ser His Ser Pro Gly Lys
435
<210> 61
<211> 19
<212> PRT
<213> artificial sequence
<220>
<223> anti-ID B-2 HC Signal sequence
<400> 61
Met Gly Trp Ser Ser Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly
1 5 10 15
Val His Ser
<210> 62
<211> 107
<212> PRT
<213> artificial sequence
<220>
<223> anti-ID B-2 VL
<400> 62
Asp Ile Gln Met Thr Gln Ser Pro Ala Ser Leu Ser Ala Ser Val Gly
1 5 10 15
Glu Thr Val Thr Ile Thr Cys Arg Ala Ser Gly Asn Ile His Asn Tyr
20 25 30
Leu Ala Trp Tyr Gln Gln Lys Gln Gly Lys Ser Pro Gln Leu Leu Val
35 40 45
Tyr Asn Ala Lys Thr Leu Ala Asp Ser Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Gln Tyr Ser Leu Lys Ile Asn Ser Leu Gln Pro
65 70 75 80
Glu Asp Phe Gly Ser Tyr Tyr Cys Gln His Phe Trp Ser Thr Pro Tyr
85 90 95
Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys
100 105
<210> 63
<211> 214
<212> PRT
<213> artificial sequence
<220>
<223> anti-ID B-2 light chain
<400> 63
Asp Ile Gln Met Thr Gln Ser Pro Ala Ser Leu Ser Ala Ser Val Gly
1 5 10 15
Glu Thr Val Thr Ile Thr Cys Arg Ala Ser Gly Asn Ile His Asn Tyr
20 25 30
Leu Ala Trp Tyr Gln Gln Lys Gln Gly Lys Ser Pro Gln Leu Leu Val
35 40 45
Tyr Asn Ala Lys Thr Leu Ala Asp Ser Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Gln Tyr Ser Leu Lys Ile Asn Ser Leu Gln Pro
65 70 75 80
Glu Asp Phe Gly Ser Tyr Tyr Cys Gln His Phe Trp Ser Thr Pro Tyr
85 90 95
Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Ala Asp Ala Ala
100 105 110
Pro Thr Val Ser Ile Phe Pro Pro Ser Ser Glu Gln Leu Thr Ser Gly
115 120 125
Gly Ala Ser Val Val Cys Phe Leu Asn Asn Phe Tyr Pro Lys Asp Ile
130 135 140
Asn Val Lys Trp Lys Ile Asp Gly Ser Glu Arg Gln Asn Gly Val Leu
145 150 155 160
Asn Ser Trp Thr Asp Gln Asp Ser Lys Asp Ser Thr Tyr Ser Met Ser
165 170 175
Ser Thr Leu Thr Leu Thr Lys Asp Glu Tyr Glu Arg His Asn Ser Tyr
180 185 190
Thr Cys Glu Ala Thr His Lys Thr Ser Thr Ser Pro Ile Val Lys Ser
195 200 205
Phe Asn Arg Asn Glu Cys
210
<210> 64
<211> 20
<212> PRT
<213> artificial sequence
<220>
<223> anti-ID B-2 LC Signal sequence
<400> 64
Met Ser Val Leu Thr Gln Val Leu Ala Leu Leu Leu Leu Trp Leu Thr
1 5 10 15
Gly Ala Arg Cys
20
<210> 65
<211> 5
<212> PRT
<213> artificial sequence
<220>
<223> anti-ID B-2 HC-CDR1
<400> 65
Arg Tyr Trp Met Asn
1 5
<210> 66
<211> 17
<212> PRT
<213> artificial sequence
<220>
<223> anti-ID B-2 HC-CDR2
<400> 66
Met Ile His Pro Ser Asp Ser Glu Thr Arg Leu Asn Gln Lys Phe Lys
1 5 10 15
Asp
<210> 67
<211> 6
<212> PRT
<213> artificial sequence
<220>
<223> anti-ID B-2 HC-CDR3
<400> 67
Ile Tyr Tyr Glu Glu Ala
1 5
<210> 68
<211> 11
<212> PRT
<213> artificial sequence
<220>
<223> anti-ID B-2 LC-CDR1
<400> 68
Arg Ala Ser Gly Asn Ile His Asn Tyr Leu Ala
1 5 10
<210> 69
<211> 7
<212> PRT
<213> artificial sequence
<220>
<223> anti-ID B-2 LC-CDR2
<400> 69
Asn Ala Lys Thr Leu Ala Asp
1 5
<210> 70
<211> 9
<212> PRT
<213> artificial sequence
<220>
<223> anti-ID B-2 LC-CDR3
<400> 70
Gln His Phe Trp Ser Thr Pro Tyr Thr
1 5
<210> 71
<211> 345
<212> DNA
<213> artificial sequence
<220>
<223> anti-ID B-2 VH
<400> 71
caggtccaac tgcagcagcc tggggctgag ctggtgaggc ctggagcttc agtgaagctg 60
tcctgcaaga cttctggcta ctccttcacc aggtactgga tgaactgggt gaagcagagg 120
cctggacaag gccttgagtg gattggcatg attcatcctt ccgatagtga aactaggtta 180
aatcagaagt tcaaggacaa ggccacattg actgtagaca attcctccag cacagcctac 240
atgcaactca gcagcccgac atctgaggac tctgcggtct attactgtgc aagcatctac 300
tatgaagagg cctggggcca agggactctg gtcactgtct ctgca 345
<210> 72
<211> 972
<212> DNA
<213> artificial sequence
<220>
<223> anti-ID B-2 CH
<400> 72
gccaaaacga cacccccatc tgtctatcca ctggcccctg gatctgctgc ccaaactaac 60
tccatggtga ccctgggatg cctggtcaag ggctatttcc ctgagccagt gacagtgacc 120
tggaactctg gatccctgtc cagcggtgtg cacaccttcc cagctgtcct gcagtctgac 180
ctctacactc tgagcagctc agtgactgtc ccctccagca cctggcccag cgagaccgtc 240
acctgcaacg ttgcccaccc ggccagcagc accaaggtgg acaagaaaat tgtgcccagg 300
gattgtggtt gtaagccttg catatgtaca gtcccagaag tatcatctgt cttcatcttc 360
cccccaaagc ccaaggatgt gctcaccatt actctgactc ctaaggtcac gtgtgttgtg 420
gtagacatca gcaaggatga tcccgaggtc cagttcagct ggtttgtaga tgatgtggag 480
gtgcacacag ctcagacgca accccgggag gagcagttca acagcacttt ccgctcagtc 540
agtgaacttc ccatcatgca ccaggactgg ctcaatggca aggagttcaa atgcagggtc 600
aacagtgcag ctttccctgc ccccatcgag aaaaccatct ccaaaaccaa aggcagaccg 660
aaggctccac aggtgtacac cattccacct cccaaggagc agatggccaa ggataaagtc 720
agtctgacct gcatgataac agacttcttc cctgaagaca ttactgtgga gtggcagtgg 780
aatgggcagc cagcggagaa ctacaagaac actcagccca tcatggacac agatggctct 840
tacttcgtct acagcaagct caatgtgcag aagagcaact gggaggcagg aaatactttc 900
acctgctctg tgttacatga gggcctgcac aaccaccata ctgagaagag cctctcccac 960
tctcctggta aa 972
<210> 73
<211> 1317
<212> DNA
<213> artificial sequence
<220>
<223> anti-ID B-2 heavy chain
<400> 73
caggtccaac tgcagcagcc tggggctgag ctggtgaggc ctggagcttc agtgaagctg 60
tcctgcaaga cttctggcta ctccttcacc aggtactgga tgaactgggt gaagcagagg 120
cctggacaag gccttgagtg gattggcatg attcatcctt ccgatagtga aactaggtta 180
aatcagaagt tcaaggacaa ggccacattg actgtagaca attcctccag cacagcctac 240
atgcaactca gcagcccgac atctgaggac tctgcggtct attactgtgc aagcatctac 300
tatgaagagg cctggggcca agggactctg gtcactgtct ctgcagccaa aacgacaccc 360
ccatctgtct atccactggc ccctggatct gctgcccaaa ctaactccat ggtgaccctg 420
ggatgcctgg tcaagggcta tttccctgag ccagtgacag tgacctggaa ctctggatcc 480
ctgtccagcg gtgtgcacac cttcccagct gtcctgcagt ctgacctcta cactctgagc 540
agctcagtga ctgtcccctc cagcacctgg cccagcgaga ccgtcacctg caacgttgcc 600
cacccggcca gcagcaccaa ggtggacaag aaaattgtgc ccagggattg tggttgtaag 660
ccttgcatat gtacagtccc agaagtatca tctgtcttca tcttcccccc aaagcccaag 720
gatgtgctca ccattactct gactcctaag gtcacgtgtg ttgtggtaga catcagcaag 780
gatgatcccg aggtccagtt cagctggttt gtagatgatg tggaggtgca cacagctcag 840
acgcaacccc gggaggagca gttcaacagc actttccgct cagtcagtga acttcccatc 900
atgcaccagg actggctcaa tggcaaggag ttcaaatgca gggtcaacag tgcagctttc 960
cctgccccca tcgagaaaac catctccaaa accaaaggca gaccgaaggc tccacaggtg 1020
tacaccattc cacctcccaa ggagcagatg gccaaggata aagtcagtct gacctgcatg 1080
ataacagact tcttccctga agacattact gtggagtggc agtggaatgg gcagccagcg 1140
gagaactaca agaacactca gcccatcatg gacacagatg gctcttactt cgtctacagc 1200
aagctcaatg tgcagaagag caactgggag gcaggaaata ctttcacctg ctctgtgtta 1260
catgagggcc tgcacaacca ccatactgag aagagcctct cccactctcc tggtaaa 1317
<210> 74
<211> 57
<212> DNA
<213> artificial sequence
<220>
<223> anti-ID B-2 HC Signal sequence
<400> 74
atgggatgga gctctatcat cctcttcttg gtagcaacag ctacaggtgt ccactcc 57
<210> 75
<211> 321
<212> DNA
<213> artificial sequence
<220>
<223> anti-ID B-2 VL
<400> 75
gacatccaga tgactcagtc tccagcctcc ctatctgcat ctgtgggaga aactgtcacc 60
atcacatgtc gagcaagtgg gaatattcac aattatttag catggtatca gcagaaacag 120
ggaaaatctc ctcagctcct ggtctataat gcaaaaacct tagcagatag tgtgccatca 180
aggttcagtg gcagtggatc aggaacacaa tattctctca agatcaacag cctgcagcct 240
gaagattttg ggagttatta ctgtcaacat ttttggagta ctccgtacac gttcggaggg 300
gggaccaagc tggaaataaa a 321
<210> 76
<211> 642
<212> DNA
<213> artificial sequence
<220>
<223> anti-ID B-2 light chain
<400> 76
gacatccaga tgactcagtc tccagcctcc ctatctgcat ctgtgggaga aactgtcacc 60
atcacatgtc gagcaagtgg gaatattcac aattatttag catggtatca gcagaaacag 120
ggaaaatctc ctcagctcct ggtctataat gcaaaaacct tagcagatag tgtgccatca 180
aggttcagtg gcagtggatc aggaacacaa tattctctca agatcaacag cctgcagcct 240
gaagattttg ggagttatta ctgtcaacat ttttggagta ctccgtacac gttcggaggg 300
gggaccaagc tggaaataaa acgggctgat gctgcaccaa ctgtatccat cttcccacca 360
tccagtgagc agttaacatc tggaggtgcc tcagtcgtgt gcttcttgaa caacttctac 420
cccaaagaca tcaatgtcaa gtggaagatt gatggcagtg aacgacaaaa tggcgtcctg 480
aacagttgga ctgatcagga cagcaaagac agcacctaca gcatgagcag caccctcacg 540
ttgaccaagg acgagtatga acgacataac agctatacct gtgaggccac tcacaagaca 600
tcaacttcac ccattgtcaa gagcttcaac aggaatgagt gt 642
<210> 77
<211> 60
<212> DNA
<213> artificial sequence
<220>
<223> anti-ID B-2 LC Signal sequence
<400> 77
atgagtgtgc tcactcaggt cctggcgttg ctgctgctgt ggcttacagg tgccagatgt 60
<210> 78
<211> 7
<212> PRT
<213> artificial sequence
<220>
<223> anti-ID HC-CDR1
<400> 78
Asp Tyr Thr Phe Thr Ser Tyr
1 5
<210> 79
<211> 10
<212> PRT
<213> artificial sequence
<220>
<223> anti-ID HC-CDR1
<400> 79
Asp Tyr Thr Phe Thr Ser Tyr Trp Met His
1 5 10
<210> 80
<211> 6
<212> PRT
<213> artificial sequence
<220>
<223> anti-ID HC-CDR1
<400> 80
Thr Ser Tyr Trp Met His
1 5
<210> 81
<211> 6
<212> PRT
<213> artificial sequence
<220>
<223> anti-ID HC-CDR2
<400> 81
Tyr Pro Gly Ser Gly Gly
1 5
<210> 82
<211> 10
<212> PRT
<213> artificial sequence
<220>
<223> anti-ID HC-CDR2
<400> 82
Asn Ile Tyr Pro Gly Ser Gly Gly Thr Asn
1 5 10
<210> 83
<211> 13
<212> PRT
<213> artificial sequence
<220>
<223> anti-ID HC-CDR2
<400> 83
Trp Ile Gly Asn Ile Tyr Pro Gly Ser Gly Gly Thr Asn
1 5 10
<210> 84
<211> 14
<212> PRT
<213> artificial sequence
<220>
<223> anti-ID HC-CDR3
<400> 84
Thr Arg Glu Val Thr Thr Val Ala Tyr Tyr Tyr Ser Met Asp
1 5 10
<210> 85
<211> 7
<212> PRT
<213> artificial sequence
<220>
<223> anti-ID LC-CDR1
<400> 85
Ser Asn Tyr Leu Asn Trp Tyr
1 5
<210> 86
<211> 10
<212> PRT
<213> artificial sequence
<220>
<223> anti-ID LC-CDR2
<400> 86
Leu Leu Ile Tyr Tyr Thr Ser Arg Leu His
1 5 10
<210> 87
<211> 8
<212> PRT
<213> artificial sequence
<220>
<223> anti-ID LC-CDR3
<400> 87
Gln Gln Gly Lys Thr Val Pro Phe
1 5
<210> 88
<211> 7
<212> PRT
<213> anti-ID B-1 HC-CDR1
<400> 88
Gly Tyr Thr Phe Thr Asp Tyr
1 5
<210> 89
<211> 10
<212> PRT
<213> artificial sequence
<220>
<223> anti-ID B-1 HC-CDR1
<400> 89
Gly Tyr Thr Phe Thr Asp Tyr Tyr Met Lys
1 5 10
<210> 90
<211> 6
<212> PRT
<213> artificial sequence
<220>
<223> anti-ID B-1 HC-CDR1
<400> 90
Thr Asp Tyr Tyr Met Lys
1 5
<210> 91
<211> 6
<212> PRT
<213> artificial sequence
<220>
<223> anti-ID B-1 HC-CDR2
<400> 91
Asn Pro Asn Asn Gly Gly
1 5
<210> 92
<211> 10
<212> PRT
<213> artificial sequence
<220>
<223> anti-ID B-1 HC-CDR2
<400> 92
Asp Ile Asn Pro Asn Asn Gly Gly Thr Asp
1 5 10
<210> 93
<211> 13
<212> PRT
<213> artificial sequence
<220>
<223> anti-ID B-1 HC-CDR2
<400> 93
Trp Ile Gly Asp Ile Asn Pro Asn Asn Gly Gly Thr Asp
1 5 10
<210> 94
<211> 14
<212> PRT
<213> artificial sequence
<220>
<223> anti-ID B-1 HC-CDR3
<400> 94
Ala Arg Glu Gly Asn Asn Tyr Gly Ser Arg Asp Ala Met Asp
1 5 10
<210> 95
<211> 6
<212> PRT
<213> artificial sequence
<220>
<223> anti-ID B-1 LC-CDR1
<400> 95
Ile Tyr Met Tyr Trp Tyr
1 5
<210> 96
<211> 10
<212> PRT
<213> artificial sequence
<220>
<223> anti-ID B-1 LC-CDR2
<400> 96
Pro Trp Ile Tyr Leu Thr Ser Asn Leu Ala
1 5 10
<210> 97
<211> 8
<212> PRT
<213> artificial sequence
<220>
<223> anti-ID B-1 LC-CDR3
<400> 97
Gln Gln Trp Ser Ser Asn Pro Leu
1 5
<210> 98
<211> 7
<212> PRT
<213> artificial sequence
<220>
<223> anti-ID B-2 HC-CDR1
<400> 98
Gly Tyr Ser Phe Thr Arg Tyr
1 5
<210> 99
<211> 10
<212> PRT
<213> artificial sequence
<220>
<223> anti-ID B-2 HC-CDR1
<400> 99
Gly Tyr Ser Phe Thr Arg Tyr Trp Met Asn
1 5 10
<210> 100
<211> 6
<212> PRT
<213> artificial sequence
<220>
<223> anti-ID B-2 HC-CDR1
<400> 100
Thr Arg Tyr Trp Met Asn
1 5
<210> 101
<211> 6
<212> PRT
<213> artificial sequence
<220>
<223> anti-ID B-2 HC-CDR2
<400> 101
His Pro Ser Asp Ser Glu
1 5
<210> 102
<211> 10
<212> PRT
<213> artificial sequence
<220>
<223> anti-ID B-2 HC-CDR2
<400> 102
Met Ile His Pro Ser Asp Ser Glu Thr Arg
1 5 10
<210> 103
<211> 13
<212> PRT
<213> artificial sequence
<220>
<223> anti-ID B-2 HC-CDR2
<400> 103
Trp Ile Gly Met Ile His Pro Ser Asp Ser Glu Thr Arg
1 5 10
<210> 104
<211> 7
<212> PRT
<213> artificial sequence
<220>
<223> anti-ID B-2 HC-CDR3
<400> 104
Ala Ser Ile Tyr Tyr Glu Glu
1 5
<210> 105
<211> 7
<212> PRT
<213> artificial sequence
<220>
<223> anti-ID B-2 LC-CDR1
<400> 105
His Asn Tyr Leu Ala Trp Tyr
1 5
<210> 106
<211> 10
<212> PRT
<213> artificial sequence
<220>
<223> anti-ID B-2 LC-CDR2
<400> 106
Leu Leu Val Tyr Asn Ala Lys Thr Leu Ala
1 5 10
<210> 107
<211> 8
<212> PRT
<213> artificial sequence
<220>
<223> anti-ID B-2 LC-CDR3
<400> 107
Gln His Phe Trp Ser Thr Pro Tyr
1 5
<210> 108
<211> 10
<212> PRT
<213> artificial sequence
<220>
<223> HC-CDR1 consensus sequences
<220>
<221> variant
<222> 3
<223> Xaa=T or S
<220>
<221> variant
<222> 5
<223> Xaa=T or S
<220>
<221> variant
<222> 6
<223> xaa=d or R
<220>
<221> variant
<222> 8
<223> xaa=y or W
<220>
<221> variant
<222> 10
<223> xaa=k or N
<400> 108
Gly Tyr Xaa Phe Xaa Xaa Tyr Xaa Met Xaa
1 5 10
<210> 109
<211> 20
<212> PRT
<213> artificial sequence
<220>
<223> HC-CDR2 consensus sequences
<220>
<221> variant
<222> 4
<223> xaa=d or M
<220>
<221> variant
<222> 6
<223> xaa=n or H
<220>
<221> variant
<222> 8
<223> Xaa=N or S
<220>
<221> variant
<222> 9
<223> xaa=n or D
<220>
<221> variant
<222> 10
<223> Xaa=G or S
<220>
<221> variant
<222> (11)...(11)
<223> xaa=g or E
<220>
<221> variant
<222> (13)...(13)
<223> xaa=d or R
<220>
<221> variant
<222> (14)...(14)
<223> xaa=y or L
<220>
<221> variant
<222> (17)...(17)
<223> xaa=n or K
<220>
<221> variant
<222> (20)...(20)
<223> xaa=g or D
<400> 109
Trp Ile Gly Xaa Ile Xaa Pro Xaa Xaa Xaa Xaa Thr Xaa Xaa Asn Gln
1 5 10 15
Xaa Phe Lys Xaa
20
<210> 110
<211> 15
<212> PRT
<213> artificial sequence
<220>
<223> HC-CDR3 consensus sequences
<220>
<221> variant
<222> 2
<223> Xaa=R or S
<220>
<221> variant
<222> 3
<223> Xaa=E or I
<220>
<221> variant
<222> 4
<223> xaa=g or Y
<220>
<221> variant
<222> 5
<223> Xaa=N or Y
<220>
<221> variant
<222> 6
<223> Xaa=N or E
<220>
<221> variant
<222> (7)...(7)
<223> xaa=y or null value
<220>
<221> variant
<222> (8)...(8)
<223> xaa=g or null value
<220>
<221> variant
<222> (9)...(9)
<223> xaa=s or null value
<220>
<221> variant
<222> (10)...(10)
<223> xaa=r or null value
<220>
<221> variant
<222> (11)...(11)
<223> xaa=d or null value
<220>
<221> variant
<222> (12)...(12)
<223> xaa=a or null value
<220>
<221> variant
<222> (13)...(13)
<223> x13=m or null value
<220>
<221> variant
<222> (14)...(14)
<223> x14=d or E
<220>
<221> variant
<222> (15)...(15)
<223> x15=y or a
<400> 110
Ala Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
1 5 10 15
<210> 111
<211> 13
<212> PRT
<213> artificial sequence
<220>
<223> LC-CDR1 consensus sequences
<220>
<221> variant
<222> (1)...(1)
<223> Xaa=S or R
<220>
<221> variant
<222> (3)...(3)
<223> Xaa=S or R
<220>
<221> variant
<222> (4)...(4)
<223> Xaa=S or G
<220>
<221> variant
<222> (5)...(5)
<223> xaa=g or N
<220>
<221> variant
<222> (6)...(6)
<223> Xaa=V or I
<220>
<221> variant
<222> (7)...(7)
<223> xaa=i or H
<220>
<221> variant
<222> (8)...(8)
<223> xaa=n or null value
<220>
<221> variant
<222> (10)...(10)
<223> xaa=m or L
<220>
<221> variant
<222> (11)...(11)
<223> xaa=y or a
<400> 111
Xaa Ala Xaa Xaa Xaa Xaa Xaa Xaa Tyr Xaa Xaa Trp Tyr
1 5 10
<210> 112
<211> 11
<212> PRT
<213> artificial sequence
<220>
<223> LC-CDR2 consensus sequences
<220>
<221> variant
<222> (1)...(1)
<223> Xaa=P or L
<220>
<221> variant
<222> (2)...(2)
<223> xaa=w or L
<220>
<221> variant
<222> (3)...(3)
<223> Xaa=I or V
<220>
<221> variant
<222> (5)...(5)
<223> Xaa=L or N
<220>
<221> variant
<222> (6)...(6)
<223> xaa=t or a
<220>
<221> variant
<222> (7)...(7)
<223> Xaa=S or K
<220>
<221> variant
<222> (8)...(8)
<223> xaa=n or T
<220>
<221> variant
<222> (11)...(11)
<223> Xaa=S or D
<400> 112
Xaa Xaa Xaa Tyr Xaa Xaa Xaa Xaa Leu Ala Xaa
1 5 10
<210> 113
<211> 9
<212> PRT
<213> artificial sequence
<220>
<223> LC-CDR3 consensus sequences
<220>
<221> variant
<222> (2)...(2)
<223> xaa=q or H
<220>
<221> variant
<222> (3)...(3)
<223> xaa=w or F
<220>
<221> variant
<222> (4)...(4)
<223> Xaa=S or W
<220>
<221> variant
<222> (5)...(5)
<223> Xaa=S or W
<220>
<221> variant
<222> (6)...(6)
<223> xaa=n or T
<220>
<221> variant
<222> (8)...(8)
<223> Xaa=L or Y
<400> 113
Gln Xaa Xaa Xaa Xaa Xaa Pro Xaa Thr
1 5
<210> 114
<211> 5
<212> PRT
<213> mice
<220>
<223> SJ25C1 HC-CDR1
<400> 114
Ser Tyr Trp Met Asn
1 5
<210> 115
<211> 17
<212> PRT
<213> mice
<220>
<223> SJ25C1 HC-CDR2
<400> 115
Gln Ile Tyr Pro Gly Asp Gly Asp Thr Asn Tyr Asn Gly Lys Phe Lys
1 5 10 15
Gly
<210> 116
<211> 13
<212> PRT
<213> mice
<220>
<223> SJ25C1 HC-CDR3
<400> 116
Lys Thr Ile Ser Ser Val Val Asp Phe Tyr Phe Asp Tyr
1 5 10
<210> 117
<211> 11
<212> PRT
<213> mice
<220>
<223> SJ25C1 LC-CDR1
<400> 117
Lys Ala Ser Gln Asn Val Gly Thr Asn Val Ala
1 5 10
<210> 118
<211> 7
<212> PRT
<213> mice
<220>
<223> SJ25C1 LC-CDR2
<400> 118
Ser Ala Thr Tyr Arg Asn Ser
1 5
<210> 119
<211> 9
<212> PRT
<213> mice
<220>
<223> SJ25C1 LC-CDR3
<400> 119
Gln Gln Tyr Asn Arg Tyr Pro Tyr Thr
1 5
<210> 120
<211> 5
<212> PRT
<213> mice
<220>
<223> SJ25C1 LC-CDR3
<400> 120
Asp Tyr Gly Val Ser
1 5
<210> 121
<211> 16
<212> PRT
<213> mice
<220>
<223> FMC63 HC-CDR2
<400> 121
Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr Asn Ser Ala Leu Lys Ser
1 5 10 15
<210> 122
<211> 12
<212> PRT
<213> mice
<220>
<223> FMC63 HC-CDR3
<400> 122
His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr
1 5 10
<210> 123
<211> 11
<212> PRT
<213> mice
<220>
<223> FMC63 LC-CDR1
<400> 123
Arg Ala Ser Gln Asp Ile Ser Lys Tyr Leu Asn
1 5 10
<210> 124
<211> 7
<212> PRT
<213> mice
<220>
<223> FMC63 LC-CDR2
<400> 124
His Thr Ser Arg Leu His Ser
1 5
<210> 125
<211> 9
<212> PRT
<213> mice
<220>
<223> FMC63 LC-CDR3
<400> 125
Gln Gln Gly Asn Thr Leu Pro Tyr Thr
1 5
<210> 126
<211> 24
<212> PRT
<213> artificial sequence
<220>
<223> T2A
<400> 126
Leu Glu Gly Gly Gly Glu Gly Arg Gly Ser Leu Leu Thr Cys Gly Asp
1 5 10 15
Val Glu Glu Asn Pro Gly Pro Arg
20
<210> 127
<211> 18
<212> PRT
<213> artificial sequence
<220>
<223> T2A
<400> 127
Glu Gly Arg Gly Ser Leu Leu Thr Cys Gly Asp Val Glu Glu Asn Pro
1 5 10 15
Gly Pro
<210> 128
<211> 22
<212> PRT
<213> artificial sequence
<220>
<223> P2A
<400> 128
Gly Ser Gly Ala Thr Asn Phe Ser Leu Leu Lys Gln Ala Gly Asp Val
1 5 10 15
Glu Glu Asn Pro Gly Pro
20
<210> 129
<211> 19
<212> PRT
<213> artificial sequence
<220>
<223> P2A
<400> 129
Ala Thr Asn Phe Ser Leu Leu Lys Gln Ala Gly Asp Val Glu Glu Asn
1 5 10 15
Pro Gly Pro
<210> 130
<211> 20
<212> PRT
<213> artificial sequence
<220>
<223> E2A
<400> 130
Gln Cys Thr Asn Tyr Ala Leu Leu Lys Leu Ala Gly Asp Val Glu Ser
1 5 10 15
Asn Pro Gly Pro
20
<210> 131
<211> 22
<212> PRT
<213> artificial sequence
<220>
<223> F2A
<400> 131
Val Lys Gln Thr Leu Asn Phe Asp Leu Leu Lys Leu Ala Gly Asp Val
1 5 10 15
Glu Ser Asn Pro Gly Pro
20
<210> 132
<211> 459
<212> PRT
<213> artificial sequence
<220>
<223> exemplary humanized anti-SJ 25C1 heavy chain
<400> 132
Gln Val Gln Leu Lys Gln Ser Gly Pro Gly Leu Val Gln Pro Ser Gln
1 5 10 15
Ser Leu Ser Ile Thr Cys Thr Val Ser Asp Tyr Thr Phe Thr Ser Tyr
20 25 30
Gly Val His Trp Val Arg Gln Ser Pro Gly Lys Gly Leu Glu Trp Leu
35 40 45
Gly Val Ile Tyr Pro Gly Ser Gly Gly Thr Asp Tyr Asn Thr Pro Phe
50 55 60
Thr Ser Arg Leu Ser Ile Asn Lys Asp Asn Ser Lys Ser Gln Val Phe
65 70 75 80
Phe Lys Met Asn Ser Leu Gln Ser Asn Asp Thr Ala Ile Tyr Tyr Cys
85 90 95
Ala Arg Glu Val Thr Thr Val Ala Tyr Tyr Tyr Ser Met Asp Tyr Trp
100 105 110
Gly Gln Gly Thr Leu Val Thr Val Ser Ala Thr Leu Val Thr Val Ser
115 120 125
Ala Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser
130 135 140
Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp
145 150 155 160
Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr
165 170 175
Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr
180 185 190
Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln
195 200 205
Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp
210 215 220
Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro
225 230 235 240
Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro
245 250 255
Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr
260 265 270
Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn
275 280 285
Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg
290 295 300
Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val
305 310 315 320
Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser
325 330 335
Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys
340 345 350
Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp
355 360 365
Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe
370 375 380
Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu
385 390 395 400
Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe
405 410 415
Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly
420 425 430
Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr
435 440 445
Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
450 455
<210> 133
<211> 214
<212> PRT
<213> artificial sequence
<220>
<223> exemplary humanized anti-SJ 25C1 light chain
<400> 133
Asp Ile Leu Leu Thr Gln Ser Pro Val Ile Leu Ser Val Ser Pro Gly
1 5 10 15
Glu Arg Val Ser Phe Ser Cys Arg Ala Ser Gln Asp Ile Ser Asn Tyr
20 25 30
Leu Asn Trp Tyr Gln Gln Arg Thr Asn Gly Ser Pro Arg Leu Leu Ile
35 40 45
Lys Tyr Thr Ser Arg Leu His Ser Gly Ile Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Ser Ile Asn Ser Val Glu Ser
65 70 75 80
Glu Asp Ile Ala Asp Tyr Tyr Cys Gln Gln Gly Lys Thr Val Pro Phe
85 90 95
Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys Arg Thr Val Ala Ala
100 105 110
Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly
115 120 125
Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala
130 135 140
Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln
145 150 155 160
Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser
165 170 175
Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr
180 185 190
Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser
195 200 205
Phe Asn Arg Gly Glu Cys
210
<210> 134
<211> 459
<212> PRT
<213> artificial sequence
<220>
<223> exemplary humanized anti-SJ 25C1 heavy chain
<400> 134
Gln Val Gln Leu Lys Gln Ser Gly Pro Gly Leu Val Gln Pro Ser Gln
1 5 10 15
Ser Leu Ser Ile Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Ser Tyr
20 25 30
Trp Met His Trp Val Arg Gln Ser Pro Gly Lys Gly Leu Glu Trp Ile
35 40 45
Gly Asn Ile Tyr Pro Gly Ser Gly Gly Thr Asn Tyr Asn Thr Pro Phe
50 55 60
Thr Ser Arg Leu Ser Ile Asn Lys Asp Asn Ser Lys Ser Gln Val Phe
65 70 75 80
Phe Lys Met Asn Ser Leu Gln Ser Asn Asp Thr Ala Ile Tyr Tyr Cys
85 90 95
Thr Arg Glu Val Thr Thr Val Ala Tyr Tyr Tyr Ser Met Asp Tyr Trp
100 105 110
Gly Gln Gly Thr Leu Val Thr Val Ser Ala Thr Leu Val Thr Val Ser
115 120 125
Ala Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser
130 135 140
Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp
145 150 155 160
Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr
165 170 175
Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr
180 185 190
Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln
195 200 205
Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp
210 215 220
Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro
225 230 235 240
Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro
245 250 255
Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr
260 265 270
Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn
275 280 285
Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg
290 295 300
Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val
305 310 315 320
Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser
325 330 335
Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys
340 345 350
Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp
355 360 365
Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe
370 375 380
Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu
385 390 395 400
Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe
405 410 415
Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly
420 425 430
Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr
435 440 445
Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
450 455
<210> 135
<211> 214
<212> PRT
<213> artificial sequence
<220>
<223> exemplary humanized anti-SJ 25C1 light chain
<400> 135
Asp Ile Leu Leu Thr Gln Ser Pro Val Ile Leu Ser Val Ser Pro Gly
1 5 10 15
Glu Arg Val Ser Phe Ser Cys Arg Ala Ser Gln Ser Ile Ser Asn Tyr
20 25 30
Leu Asn Trp Tyr Gln Gln Arg Thr Asn Gly Ser Pro Arg Leu Leu Ile
35 40 45
Tyr Tyr Thr Ser Arg Leu His Ser Gly Ile Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Ser Ile Asn Ser Val Glu Ser
65 70 75 80
Glu Asp Ile Ala Asp Tyr Tyr Cys Gln Gln Gly Lys Thr Val Pro Phe
85 90 95
Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys Arg Thr Val Ala Ala
100 105 110
Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly
115 120 125
Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala
130 135 140
Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln
145 150 155 160
Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser
165 170 175
Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr
180 185 190
Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser
195 200 205
Phe Asn Arg Gly Glu Cys
210
<210> 136
<211> 459
<212> PRT
<213> artificial sequence
<220>
<223> exemplary humanized anti-SJ 25C1 heavy chain
<400> 136
Gln Val Gln Leu Lys Gln Ser Gly Pro Gly Leu Val Gln Pro Ser Gln
1 5 10 15
Ser Leu Ser Ile Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Ser Tyr
20 25 30
Trp Met His Trp Val Arg Gln Ser Pro Gly Lys Gly Leu Glu Trp Leu
35 40 45
Gly Asn Ile Tyr Pro Gly Ser Gly Gly Thr Asn Tyr Asp Glu Lys Phe
50 55 60
Lys Arg Arg Leu Ser Ile Asn Lys Asp Asn Ser Lys Ser Gln Val Phe
65 70 75 80
Phe Lys Met Asn Ser Leu Gln Ser Asn Asp Thr Ala Ile Tyr Tyr Cys
85 90 95
Ala Arg Glu Val Thr Thr Val Ala Tyr Tyr Tyr Ser Met Asp Tyr Trp
100 105 110
Gly Gln Gly Thr Leu Val Thr Val Ser Ala Thr Leu Val Thr Val Ser
115 120 125
Ala Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser
130 135 140
Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp
145 150 155 160
Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr
165 170 175
Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr
180 185 190
Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln
195 200 205
Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp
210 215 220
Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro
225 230 235 240
Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro
245 250 255
Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr
260 265 270
Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn
275 280 285
Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg
290 295 300
Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val
305 310 315 320
Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser
325 330 335
Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys
340 345 350
Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp
355 360 365
Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe
370 375 380
Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu
385 390 395 400
Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe
405 410 415
Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly
420 425 430
Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr
435 440 445
Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
450 455
<210> 137
<211> 214
<212> PRT
<213> artificial sequence
<220>
<223> exemplary humanized anti-SJ 25C1 light chain
<400> 137
Asp Ile Leu Leu Thr Gln Ser Pro Val Ile Leu Ser Val Ser Pro Gly
1 5 10 15
Glu Arg Val Ser Phe Ser Cys Arg Ala Ser Gln Asp Ile Ser Asn Tyr
20 25 30
Leu Asn Trp Tyr Gln Gln Arg Thr Asn Gly Ser Pro Arg Leu Leu Ile
35 40 45
Lys Tyr Thr Ser Arg Leu His Ser Gly Ile Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Ser Ile Asn Ser Val Glu Ser
65 70 75 80
Glu Asp Ile Ala Asp Tyr Tyr Cys Gln Gln Gly Lys Thr Val Pro Phe
85 90 95
Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys Arg Thr Val Ala Ala
100 105 110
Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly
115 120 125
Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala
130 135 140
Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln
145 150 155 160
Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser
165 170 175
Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr
180 185 190
Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser
195 200 205
Phe Asn Arg Gly Glu Cys
210
<210> 138
<211> 459
<212> PRT
<213> artificial sequence
<220>
<223> exemplary humanized anti-FMC 63 heavy chain
<400> 138
Gln Val Gln Leu Lys Gln Ser Gly Pro Gly Leu Val Gln Pro Ser Gln
1 5 10 15
Ser Leu Ser Ile Thr Cys Thr Val Ser Gly Tyr Thr Phe Thr Asp Tyr
20 25 30
Gly Val His Trp Val Arg Gln Ser Pro Gly Lys Gly Leu Glu Trp Leu
35 40 45
Gly Val Ile Asn Pro Asn Asn Gly Gly Thr Asp Tyr Asn Thr Pro Phe
50 55 60
Thr Ser Arg Leu Ser Ile Asn Lys Asp Asn Ser Lys Ser Gln Val Phe
65 70 75 80
Phe Lys Met Asn Ser Leu Gln Ser Asn Asp Thr Ala Ile Tyr Tyr Cys
85 90 95
Ala Arg Glu Gly Asn Asn Tyr Gly Ser Arg Asp Ala Met Asp Tyr Trp
100 105 110
Gly Gln Gly Thr Leu Val Thr Val Ser Ala Thr Leu Val Thr Val Ser
115 120 125
Ala Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser
130 135 140
Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp
145 150 155 160
Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr
165 170 175
Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr
180 185 190
Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln
195 200 205
Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp
210 215 220
Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro
225 230 235 240
Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro
245 250 255
Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr
260 265 270
Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn
275 280 285
Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg
290 295 300
Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val
305 310 315 320
Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser
325 330 335
Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys
340 345 350
Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp
355 360 365
Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe
370 375 380
Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu
385 390 395 400
Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe
405 410 415
Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly
420 425 430
Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr
435 440 445
Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
450 455
<210> 139
<211> 213
<212> PRT
<213> artificial sequence
<220>
<223> exemplary humanized anti-FMC 63 light chain
<400> 139
Asp Ile Leu Leu Thr Gln Ser Pro Val Ile Leu Ser Val Ser Pro Gly
1 5 10 15
Glu Arg Val Ser Phe Ser Cys Ser Ala Ser Ser Gly Val Ile Tyr Met
20 25 30
Tyr Trp Tyr Gln Gln Arg Thr Asn Gly Ser Pro Arg Leu Leu Ile Lys
35 40 45
Leu Thr Ser Asn Leu Ala Ser Gly Ile Pro Ser Arg Phe Ser Gly Ser
50 55 60
Gly Ser Gly Thr Asp Phe Thr Leu Ser Ile Asn Ser Val Glu Ser Glu
65 70 75 80
Asp Ile Ala Asp Tyr Tyr Cys Gln Gln Trp Ser Ser Asn Pro Leu Thr
85 90 95
Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys Arg Thr Val Ala Ala Pro
100 105 110
Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr
115 120 125
Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys
130 135 140
Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu
145 150 155 160
Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser
165 170 175
Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala
180 185 190
Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe
195 200 205
Asn Arg Gly Glu Cys
210
<210> 140
<211> 459
<212> PRT
<213> artificial sequence
<220>
<223> exemplary humanized anti-FMC 63 heavy chain
<400> 140
Gln Val Gln Leu Lys Gln Ser Gly Pro Gly Leu Val Gln Pro Ser Gln
1 5 10 15
Ser Leu Ser Ile Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Asp Tyr
20 25 30
Tyr Met Lys Trp Val Arg Gln Ser Pro Gly Lys Gly Leu Glu Trp Leu
35 40 45
Gly Asp Ile Asn Pro Asn Asn Gly Gly Thr Asp Tyr Asn Gln Asn Phe
50 55 60
Lys Gly Arg Leu Ser Ile Asn Lys Asp Asn Ser Lys Ser Gln Val Phe
65 70 75 80
Phe Lys Met Asn Ser Leu Gln Ser Asn Asp Thr Ala Ile Tyr Tyr Cys
85 90 95
Ala Arg Glu Gly Asn Asn Tyr Gly Ser Arg Asp Ala Met Asp Tyr Trp
100 105 110
Gly Gln Gly Thr Leu Val Thr Val Ser Ala Thr Leu Val Thr Val Ser
115 120 125
Ala Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser
130 135 140
Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp
145 150 155 160
Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr
165 170 175
Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr
180 185 190
Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln
195 200 205
Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp
210 215 220
Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro
225 230 235 240
Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro
245 250 255
Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr
260 265 270
Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn
275 280 285
Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg
290 295 300
Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val
305 310 315 320
Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser
325 330 335
Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys
340 345 350
Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp
355 360 365
Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe
370 375 380
Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu
385 390 395 400
Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe
405 410 415
Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly
420 425 430
Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr
435 440 445
Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
450 455
<210> 141
<211> 213
<212> PRT
<213> artificial sequence
<220>
<223> exemplary humanized anti-FMC 63 light chain
<400> 141
Asp Ile Leu Leu Thr Gln Ser Pro Val Ile Leu Ser Val Ser Pro Gly
1 5 10 15
Glu Arg Val Ser Phe Ser Cys Ser Ala Ser Ser Gly Val Ile Tyr Met
20 25 30
Tyr Trp Tyr Gln Gln Arg Thr Asn Gly Ser Pro Arg Leu Leu Ile Lys
35 40 45
Leu Thr Ser Asn Leu Ala Ser Gly Ile Pro Ser Arg Phe Ser Gly Ser
50 55 60
Gly Ser Gly Thr Asp Phe Thr Leu Ser Ile Asn Ser Val Glu Ser Glu
65 70 75 80
Asp Ile Ala Asp Tyr Tyr Cys Gln Gln Trp Ser Ser Asn Pro Leu Thr
85 90 95
Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys Arg Thr Val Ala Ala Pro
100 105 110
Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr
115 120 125
Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys
130 135 140
Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu
145 150 155 160
Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser
165 170 175
Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala
180 185 190
Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe
195 200 205
Asn Arg Gly Glu Cys
210
<210> 142
<211> 459
<212> PRT
<213> artificial sequence
<220>
<223> exemplary humanized anti-FMC 63 heavy chain
<400> 142
Gln Val Gln Leu Lys Gln Ser Gly Pro Gly Leu Val Gln Pro Ser Gln
1 5 10 15
Ser Leu Ser Ile Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Asp Tyr
20 25 30
Tyr Met Lys Trp Val Arg Gln Ser Pro Gly Lys Gly Leu Glu Trp Ile
35 40 45
Gly Asp Ile Asn Pro Asn Asn Gly Gly Thr Asp Tyr Asn Thr Pro Phe
50 55 60
Thr Ser Arg Leu Ser Ile Asn Lys Asp Asn Ser Lys Ser Gln Val Phe
65 70 75 80
Phe Lys Met Asn Ser Leu Gln Ser Asn Asp Thr Ala Ile Tyr Tyr Cys
85 90 95
Ala Arg Glu Gly Asn Asn Tyr Gly Ser Arg Asp Ala Met Asp Tyr Trp
100 105 110
Gly Gln Gly Thr Leu Val Thr Val Ser Ala Thr Leu Val Thr Val Ser
115 120 125
Ala Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser
130 135 140
Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp
145 150 155 160
Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr
165 170 175
Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr
180 185 190
Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln
195 200 205
Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp
210 215 220
Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro
225 230 235 240
Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro
245 250 255
Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr
260 265 270
Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn
275 280 285
Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg
290 295 300
Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val
305 310 315 320
Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser
325 330 335
Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys
340 345 350
Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp
355 360 365
Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe
370 375 380
Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu
385 390 395 400
Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe
405 410 415
Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly
420 425 430
Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr
435 440 445
Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
450 455
<210> 143
<211> 213
<212> PRT
<213> artificial sequence
<220>
<223> exemplary humanized anti-FMC 63 light chain
<400> 143
Asp Ile Leu Leu Thr Gln Ser Pro Val Ile Leu Ser Val Ser Pro Gly
1 5 10 15
Glu Arg Val Ser Phe Ser Cys Arg Ala Ser Gln Ser Ile Ile Tyr Met
20 25 30
Tyr Trp Tyr Gln Gln Arg Thr Asn Gly Ser Pro Arg Pro Trp Ile Tyr
35 40 45
Leu Thr Ser Asn Leu Ala Ser Gly Ile Pro Ser Arg Phe Ser Gly Ser
50 55 60
Gly Ser Gly Thr Asp Phe Thr Leu Ser Ile Asn Ser Val Glu Ser Glu
65 70 75 80
Asp Ile Ala Asp Tyr Tyr Cys Gln Gln Trp Ser Ser Asn Pro Leu Thr
85 90 95
Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys Arg Thr Val Ala Ala Pro
100 105 110
Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr
115 120 125
Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys
130 135 140
Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu
145 150 155 160
Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser
165 170 175
Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala
180 185 190
Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe
195 200 205
Asn Arg Gly Glu Cys
210
<210> 144
<211> 452
<212> PRT
<213> artificial sequence
<220>
<223> exemplary humanized anti-FMC 63 heavy chain
<400> 144
Gln Val Gln Leu Lys Gln Ser Gly Pro Gly Leu Val Gln Pro Ser Gln
1 5 10 15
Ser Leu Ser Ile Thr Cys Thr Val Ser Gly Tyr Ser Phe Thr Arg Tyr
20 25 30
Gly Val His Trp Val Arg Gln Ser Pro Gly Lys Gly Leu Glu Trp Leu
35 40 45
Gly Val Ile His Pro Ser Asp Ser Glu Thr Asp Tyr Asn Thr Pro Phe
50 55 60
Thr Ser Arg Leu Ser Ile Asn Lys Asp Asn Ser Lys Ser Gln Val Phe
65 70 75 80
Phe Lys Met Asn Ser Leu Gln Ser Asn Asp Thr Ala Ile Tyr Tyr Cys
85 90 95
Ala Arg Ile Tyr Tyr Glu Glu Ala Trp Gly Gln Gly Thr Leu Val Thr
100 105 110
Val Ser Ala Thr Leu Val Thr Val Ser Ala Ala Ser Thr Lys Gly Pro
115 120 125
Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr
130 135 140
Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr
145 150 155 160
Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro
165 170 175
Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr
180 185 190
Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn
195 200 205
His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser
210 215 220
Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu
225 230 235 240
Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu
245 250 255
Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser
260 265 270
His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu
275 280 285
Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr
290 295 300
Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn
305 310 315 320
Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro
325 330 335
Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln
340 345 350
Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val
355 360 365
Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val
370 375 380
Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro
385 390 395 400
Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr
405 410 415
Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val
420 425 430
Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu
435 440 445
Ser Pro Gly Lys
450
<210> 145
<211> 214
<212> PRT
<213> artificial sequence
<220>
<223> exemplary humanized anti-FMC 63 light chain
<400> 145
Asp Ile Leu Leu Thr Gln Ser Pro Val Ile Leu Ser Val Ser Pro Gly
1 5 10 15
Glu Arg Val Ser Phe Ser Cys Arg Ala Ser Gly Asn Ile His Asn Tyr
20 25 30
Leu Ala Trp Tyr Gln Gln Arg Thr Asn Gly Ser Pro Arg Leu Leu Ile
35 40 45
Lys Asn Ala Lys Thr Leu Ala Asp Gly Ile Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Ser Ile Asn Ser Val Glu Ser
65 70 75 80
Glu Asp Ile Ala Asp Tyr Tyr Cys Gln His Phe Trp Ser Thr Pro Tyr
85 90 95
Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys Arg Thr Val Ala Ala
100 105 110
Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly
115 120 125
Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala
130 135 140
Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln
145 150 155 160
Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser
165 170 175
Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr
180 185 190
Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser
195 200 205
Phe Asn Arg Gly Glu Cys
210
<210> 146
<211> 452
<212> PRT
<213> artificial sequence
<220>
<223> exemplary humanized anti-FMC 63 heavy chain
<400> 146
Gln Val Gln Leu Lys Gln Ser Gly Pro Gly Leu Val Gln Pro Ser Gln
1 5 10 15
Ser Leu Ser Ile Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Arg Tyr
20 25 30
Trp Met Asn Trp Val Arg Gln Ser Pro Gly Lys Gly Leu Glu Trp Leu
35 40 45
Gly Met Ile His Pro Ser Asp Ser Glu Thr Arg Leu Asn Gln Lys Phe
50 55 60
Lys Asp Arg Leu Ser Ile Asn Lys Asp Asn Ser Lys Ser Gln Val Phe
65 70 75 80
Phe Lys Met Asn Ser Leu Gln Ser Asn Asp Thr Ala Ile Tyr Tyr Cys
85 90 95
Ala Arg Ile Tyr Tyr Glu Glu Ala Trp Gly Gln Gly Thr Leu Val Thr
100 105 110
Val Ser Ala Thr Leu Val Thr Val Ser Ala Ala Ser Thr Lys Gly Pro
115 120 125
Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr
130 135 140
Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr
145 150 155 160
Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro
165 170 175
Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr
180 185 190
Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn
195 200 205
His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser
210 215 220
Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu
225 230 235 240
Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu
245 250 255
Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser
260 265 270
His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu
275 280 285
Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr
290 295 300
Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn
305 310 315 320
Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro
325 330 335
Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln
340 345 350
Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val
355 360 365
Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val
370 375 380
Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro
385 390 395 400
Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr
405 410 415
Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val
420 425 430
Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu
435 440 445
Ser Pro Gly Lys
450
<210> 147
<211> 214
<212> PRT
<213> artificial sequence
<220>
<223> exemplary humanized anti-FMC 63 light chain
<400> 147
Asp Ile Leu Leu Thr Gln Ser Pro Val Ile Leu Ser Val Ser Pro Gly
1 5 10 15
Glu Arg Val Ser Phe Ser Cys Arg Ala Ser Gly Asn Ile His Asn Tyr
20 25 30
Leu Ala Trp Tyr Gln Gln Arg Thr Asn Gly Ser Pro Arg Leu Leu Ile
35 40 45
Lys Asn Ala Lys Thr Leu Ala Asp Gly Ile Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Ser Ile Asn Ser Val Glu Ser
65 70 75 80
Glu Asp Ile Ala Asp Tyr Tyr Cys Gln His Phe Trp Ser Thr Pro Tyr
85 90 95
Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys Arg Thr Val Ala Ala
100 105 110
Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly
115 120 125
Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala
130 135 140
Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln
145 150 155 160
Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser
165 170 175
Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr
180 185 190
Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser
195 200 205
Phe Asn Arg Gly Glu Cys
210
<210> 148
<211> 452
<212> PRT
<213> artificial sequence
<220>
<223> exemplary humanized anti-FMC 63 heavy chain
<400> 148
Gln Val Gln Leu Lys Gln Ser Gly Pro Gly Leu Val Gln Pro Ser Gln
1 5 10 15
Ser Leu Ser Ile Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Arg Tyr
20 25 30
Trp Met Asn Trp Val Arg Gln Ser Pro Gly Lys Gly Leu Glu Trp Ile
35 40 45
Gly Met Ile His Pro Ser Asp Ser Glu Thr Arg Tyr Asn Thr Pro Phe
50 55 60
Thr Ser Arg Leu Ser Ile Asn Lys Asp Asn Ser Lys Ser Gln Val Phe
65 70 75 80
Phe Lys Met Asn Ser Leu Gln Ser Asn Asp Thr Ala Ile Tyr Tyr Cys
85 90 95
Ala Ser Ile Tyr Tyr Glu Glu Tyr Trp Gly Gln Gly Thr Leu Val Thr
100 105 110
Val Ser Ala Thr Leu Val Thr Val Ser Ala Ala Ser Thr Lys Gly Pro
115 120 125
Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr
130 135 140
Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr
145 150 155 160
Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro
165 170 175
Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr
180 185 190
Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn
195 200 205
His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser
210 215 220
Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu
225 230 235 240
Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu
245 250 255
Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser
260 265 270
His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu
275 280 285
Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr
290 295 300
Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn
305 310 315 320
Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro
325 330 335
Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln
340 345 350
Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val
355 360 365
Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val
370 375 380
Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro
385 390 395 400
Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr
405 410 415
Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val
420 425 430
Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu
435 440 445
Ser Pro Gly Lys
450
<210> 149
<211> 214
<212> PRT
<213> artificial sequence
<220>
<223> exemplary humanized anti-FMC 63 light chain
<400> 149
Asp Ile Leu Leu Thr Gln Ser Pro Val Ile Leu Ser Val Ser Pro Gly
1 5 10 15
Glu Arg Val Ser Phe Ser Cys Arg Ala Ser Gln Ser Ile His Asn Tyr
20 25 30
Leu Ala Trp Tyr Gln Gln Arg Thr Asn Gly Ser Pro Arg Leu Leu Val
35 40 45
Tyr Asn Ala Lys Thr Leu Ala Ser Gly Ile Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Ser Ile Asn Ser Val Glu Ser
65 70 75 80
Glu Asp Ile Ala Asp Tyr Tyr Cys Gln His Phe Trp Ser Thr Pro Tyr
85 90 95
Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys Arg Thr Val Ala Ala
100 105 110
Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly
115 120 125
Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala
130 135 140
Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln
145 150 155 160
Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser
165 170 175
Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr
180 185 190
Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser
195 200 205
Phe Asn Arg Gly Glu Cys
210
<210> 150
<211> 12
<212> PRT
<213> Chile person
<220>
<223> spacer (IgG 4 hinge) (aa)
<400> 150
Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro
1 5 10
<210> 151
<211> 36
<212> DNA
<213> Chile person
<220>
<223> spacer (IgG 4 hinge) (nt)
<400> 151
gaatctaagt acggaccgcc ctgcccccct tgccct 36
<210> 152
<211> 119
<212> PRT
<213> Chile person
<220>
<223> hinge-CH 3 spacer
<400> 152
Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Gly Gln Pro Arg
1 5 10 15
Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys
20 25 30
Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp
35 40 45
Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys
50 55 60
Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser
65 70 75 80
Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser
85 90 95
Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser
100 105 110
Leu Ser Leu Ser Leu Gly Lys
115
<210> 153
<211> 229
<212> PRT
<213> Chile person
<220>
<223> hinge-CH 2-CH3
<400> 153
Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe
1 5 10 15
Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr
20 25 30
Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val
35 40 45
Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val
50 55 60
Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser
65 70 75 80
Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu
85 90 95
Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser
100 105 110
Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro
115 120 125
Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln
130 135 140
Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala
145 150 155 160
Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr
165 170 175
Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu
180 185 190
Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser
195 200 205
Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser
210 215 220
Leu Ser Leu Gly Lys
225
<210> 154
<211> 282
<212> PRT
<213> Chile person
<220>
<223> IgD-hinge-Fc
<400> 154
Arg Trp Pro Glu Ser Pro Lys Ala Gln Ala Ser Ser Val Pro Thr Ala
1 5 10 15
Gln Pro Gln Ala Glu Gly Ser Leu Ala Lys Ala Thr Thr Ala Pro Ala
20 25 30
Thr Thr Arg Asn Thr Gly Arg Gly Gly Glu Glu Lys Lys Lys Glu Lys
35 40 45
Glu Lys Glu Glu Gln Glu Glu Arg Glu Thr Lys Thr Pro Glu Cys Pro
50 55 60
Ser His Thr Gln Pro Leu Gly Val Tyr Leu Leu Thr Pro Ala Val Gln
65 70 75 80
Asp Leu Trp Leu Arg Asp Lys Ala Thr Phe Thr Cys Phe Val Val Gly
85 90 95
Ser Asp Leu Lys Asp Ala His Leu Thr Trp Glu Val Ala Gly Lys Val
100 105 110
Pro Thr Gly Gly Val Glu Glu Gly Leu Leu Glu Arg His Ser Asn Gly
115 120 125
Ser Gln Ser Gln His Ser Arg Leu Thr Leu Pro Arg Ser Leu Trp Asn
130 135 140
Ala Gly Thr Ser Val Thr Cys Thr Leu Asn His Pro Ser Leu Pro Pro
145 150 155 160
Gln Arg Leu Met Ala Leu Arg Glu Pro Ala Ala Gln Ala Pro Val Lys
165 170 175
Leu Ser Leu Asn Leu Leu Ala Ser Ser Asp Pro Pro Glu Ala Ala Ser
180 185 190
Trp Leu Leu Cys Glu Val Ser Gly Phe Ser Pro Pro Asn Ile Leu Leu
195 200 205
Met Trp Leu Glu Asp Gln Arg Glu Val Asn Thr Ser Gly Phe Ala Pro
210 215 220
Ala Arg Pro Pro Pro Gln Pro Gly Ser Thr Thr Phe Trp Ala Trp Ser
225 230 235 240
Val Leu Arg Val Pro Ala Pro Pro Ser Pro Gln Pro Ala Thr Tyr Thr
245 250 255
Cys Val Val Ser His Glu Asp Ser Arg Thr Leu Leu Asn Ala Ser Arg
260 265 270
Ser Leu Glu Val Ser Tyr Val Thr Asp His
275 280
<210> 155
<211> 335
<212> PRT
<213> artificial sequence
<220>
<223> tEGFR
<400> 155
Arg Lys Val Cys Asn Gly Ile Gly Ile Gly Glu Phe Lys Asp Ser Leu
1 5 10 15
Ser Ile Asn Ala Thr Asn Ile Lys His Phe Lys Asn Cys Thr Ser Ile
20 25 30
Ser Gly Asp Leu His Ile Leu Pro Val Ala Phe Arg Gly Asp Ser Phe
35 40 45
Thr His Thr Pro Pro Leu Asp Pro Gln Glu Leu Asp Ile Leu Lys Thr
50 55 60
Val Lys Glu Ile Thr Gly Phe Leu Leu Ile Gln Ala Trp Pro Glu Asn
65 70 75 80
Arg Thr Asp Leu His Ala Phe Glu Asn Leu Glu Ile Ile Arg Gly Arg
85 90 95
Thr Lys Gln His Gly Gln Phe Ser Leu Ala Val Val Ser Leu Asn Ile
100 105 110
Thr Ser Leu Gly Leu Arg Ser Leu Lys Glu Ile Ser Asp Gly Asp Val
115 120 125
Ile Ile Ser Gly Asn Lys Asn Leu Cys Tyr Ala Asn Thr Ile Asn Trp
130 135 140
Lys Lys Leu Phe Gly Thr Ser Gly Gln Lys Thr Lys Ile Ile Ser Asn
145 150 155 160
Arg Gly Glu Asn Ser Cys Lys Ala Thr Gly Gln Val Cys His Ala Leu
165 170 175
Cys Ser Pro Glu Gly Cys Trp Gly Pro Glu Pro Arg Asp Cys Val Ser
180 185 190
Cys Arg Asn Val Ser Arg Gly Arg Glu Cys Val Asp Lys Cys Asn Leu
195 200 205
Leu Glu Gly Glu Pro Arg Glu Phe Val Glu Asn Ser Glu Cys Ile Gln
210 215 220
Cys His Pro Glu Cys Leu Pro Gln Ala Met Asn Ile Thr Cys Thr Gly
225 230 235 240
Arg Gly Pro Asp Asn Cys Ile Gln Cys Ala His Tyr Ile Asp Gly Pro
245 250 255
His Cys Val Lys Thr Cys Pro Ala Gly Val Met Gly Glu Asn Asn Thr
260 265 270
Leu Val Trp Lys Tyr Ala Asp Ala Gly His Val Cys His Leu Cys His
275 280 285
Pro Asn Cys Thr Tyr Gly Cys Thr Gly Pro Gly Leu Glu Gly Cys Pro
290 295 300
Thr Asn Gly Pro Lys Ile Pro Ser Ile Ala Thr Gly Met Val Gly Ala
305 310 315 320
Leu Leu Leu Leu Leu Val Val Ala Leu Gly Ile Gly Leu Phe Met
325 330 335
<210> 156
<211> 357
<212> PRT
<213> artificial sequence
<220>
<223> tEGFR
<400> 156
Met Leu Leu Leu Val Thr Ser Leu Leu Leu Cys Glu Leu Pro His Pro
1 5 10 15
Ala Phe Leu Leu Ile Pro Arg Lys Val Cys Asn Gly Ile Gly Ile Gly
20 25 30
Glu Phe Lys Asp Ser Leu Ser Ile Asn Ala Thr Asn Ile Lys His Phe
35 40 45
Lys Asn Cys Thr Ser Ile Ser Gly Asp Leu His Ile Leu Pro Val Ala
50 55 60
Phe Arg Gly Asp Ser Phe Thr His Thr Pro Pro Leu Asp Pro Gln Glu
65 70 75 80
Leu Asp Ile Leu Lys Thr Val Lys Glu Ile Thr Gly Phe Leu Leu Ile
85 90 95
Gln Ala Trp Pro Glu Asn Arg Thr Asp Leu His Ala Phe Glu Asn Leu
100 105 110
Glu Ile Ile Arg Gly Arg Thr Lys Gln His Gly Gln Phe Ser Leu Ala
115 120 125
Val Val Ser Leu Asn Ile Thr Ser Leu Gly Leu Arg Ser Leu Lys Glu
130 135 140
Ile Ser Asp Gly Asp Val Ile Ile Ser Gly Asn Lys Asn Leu Cys Tyr
145 150 155 160
Ala Asn Thr Ile Asn Trp Lys Lys Leu Phe Gly Thr Ser Gly Gln Lys
165 170 175
Thr Lys Ile Ile Ser Asn Arg Gly Glu Asn Ser Cys Lys Ala Thr Gly
180 185 190
Gln Val Cys His Ala Leu Cys Ser Pro Glu Gly Cys Trp Gly Pro Glu
195 200 205
Pro Arg Asp Cys Val Ser Cys Arg Asn Val Ser Arg Gly Arg Glu Cys
210 215 220
Val Asp Lys Cys Asn Leu Leu Glu Gly Glu Pro Arg Glu Phe Val Glu
225 230 235 240
Asn Ser Glu Cys Ile Gln Cys His Pro Glu Cys Leu Pro Gln Ala Met
245 250 255
Asn Ile Thr Cys Thr Gly Arg Gly Pro Asp Asn Cys Ile Gln Cys Ala
260 265 270
His Tyr Ile Asp Gly Pro His Cys Val Lys Thr Cys Pro Ala Gly Val
275 280 285
Met Gly Glu Asn Asn Thr Leu Val Trp Lys Tyr Ala Asp Ala Gly His
290 295 300
Val Cys His Leu Cys His Pro Asn Cys Thr Tyr Gly Cys Thr Gly Pro
305 310 315 320
Gly Leu Glu Gly Cys Pro Thr Asn Gly Pro Lys Ile Pro Ser Ile Ala
325 330 335
Thr Gly Met Val Gly Ala Leu Leu Leu Leu Leu Val Val Ala Leu Gly
340 345 350
Ile Gly Leu Phe Met
355
<210> 157
<211> 27
<212> PRT
<213> Chile person
<220>
<223> CD28 (amino acids 153-179 of accession number P10747)
<400> 157
Phe Trp Val Leu Val Val Val Gly Gly Val Leu Ala Cys Tyr Ser Leu
1 5 10 15
Leu Val Thr Val Ala Phe Ile Ile Phe Trp Val
20 25
<210> 158
<211> 66
<212> PRT
<213> Chile person
<220>
<223> CD28 (amino acids 114-179 of accession number P10747)
<400> 158
Ile Glu Val Met Tyr Pro Pro Pro Tyr Leu Asp Asn Glu Lys Ser Asn
1 5 10 15
Gly Thr Ile Ile His Val Lys Gly Lys His Leu Cys Pro Ser Pro Leu
20 25 30
Phe Pro Gly Pro Ser Lys Pro Phe Trp Val Leu Val Val Val Gly Gly
35 40 45
Val Leu Ala Cys Tyr Ser Leu Leu Val Thr Val Ala Phe Ile Ile Phe
50 55 60
Trp Val
65
<210> 159
<211> 41
<212> PRT
<213> Chile person
<220>
<223> CD28 (amino acids 180-220 of P10747)
<400> 159
Arg Ser Lys Arg Ser Arg Leu Leu His Ser Asp Tyr Met Asn Met Thr
1 5 10 15
Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro
20 25 30
Pro Arg Asp Phe Ala Ala Tyr Arg Ser
35 40
<210> 160
<211> 41
<212> PRT
<213> Chile person
<220>
<223> CD28 (LL to GG)
<400> 160
Arg Ser Lys Arg Ser Arg Gly Gly His Ser Asp Tyr Met Asn Met Thr
1 5 10 15
Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro
20 25 30
Pro Arg Asp Phe Ala Ala Tyr Arg Ser
35 40
<210> 161
<211> 42
<212> PRT
<213> Chile person
<220>
<223> 4-1BB (amino acids 214-255 of Q07011.1)
<400> 161
Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met
1 5 10 15
Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe
20 25 30
Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu
35 40
<210> 162
<211> 112
<212> PRT
<213> Chile person
<220>
<223> CD3ζ
<400> 162
Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly
1 5 10 15
Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr
20 25 30
Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys
35 40 45
Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys
50 55 60
Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg
65 70 75 80
Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala
85 90 95
Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg
100 105 110
<210> 163
<211> 112
<212> PRT
<213> Chile person
<220>
<223> CD3ζ
<400> 163
Arg Val Lys Phe Ser Arg Ser Ala Glu Pro Pro Ala Tyr Gln Gln Gly
1 5 10 15
Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr
20 25 30
Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys
35 40 45
Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys
50 55 60
Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg
65 70 75 80
Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala
85 90 95
Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg
100 105 110
<210> 164
<211> 112
<212> PRT
<213> Chile person
<220>
<223> CD3ζ
<400> 164
Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Lys Gln Gly
1 5 10 15
Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr
20 25 30
Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys
35 40 45
Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys
50 55 60
Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg
65 70 75 80
Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala
85 90 95
Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg
100 105 110

Claims (37)

1. An anti-idiotype antibody or antigen-binding fragment thereof comprising a heavy chain Variable (VH) region and a light chain Variable (VL) region, wherein
(a) The VH region comprises CDR-H1, CDR-H2 and CDR-H3, the CDR-H1, CDR-H2 and CDR-H3 being as defined in amino acid sequences SEQ ID NO: 44. 45 and 46; and the VL region comprises CDR-L1, CDR-L2 and CDR-L3, said CDR-L1, CDR-L2 and CDR-L3 being as defined in amino acid sequences SEQ ID NO: 47. 48 and 49, wherein said sequence is obtained from Kabat numbering; or (b)
(b) The VH region comprises CDR-H1, CDR-H2 and CDR-H3, the CDR-H1, CDR-H2 and CDR-H3 being as defined in amino acid sequences SEQ ID NO: 88. 91 and 46; and the VL region comprises CDR-L1, CDR-L2 and CDR-L3, said CDR-L1, CDR-L2 and CDR-L3 being as defined in amino acid sequences SEQ ID NO: 47. 48 and 49, wherein the sequence is obtained from Chothia numbering; or (b)
(c) The VH region comprises CDR-H1, CDR-H2 and CDR-H3, the CDR-H1, CDR-H2 and CDR-H3 being as defined in amino acid sequences SEQ ID NO: 89. 92 and 46; and the VL region comprises CDR-L1, CDR-L2 and CDR-L3, said CDR-L1, CDR-L2 and CDR-L3 being as defined in amino acid sequences SEQ ID NO: 47. 48 and 49, wherein the sequences are obtained from AbM numbering; or (b)
(d) The VH region comprises CDR-H1, CDR-H2 and CDR-H3, the CDR-H1, CDR-H2 and CDR-H3 being as defined in amino acid sequences SEQ ID NO: 90. 93 and 94; and the VL region comprises CDR-L1, CDR-L2 and CDR-L3, said CDR-L1, CDR-L2 and CDR-L3 being as defined in amino acid sequences SEQ ID NO: 95. 96 and 97, wherein the sequence is obtained from a Contact number; and is also provided with
Wherein the antibody or antigen-binding fragment thereof binds to a target antibody or antigen-binding fragment thereof, the target antibody being antibody FMC63, wherein the target antibody or antigen-binding fragment thereof comprises the amino acid sequence of SEQ ID NO:30 and the heavy chain variable region set forth in SEQ ID NO: 31.
2. The anti-idiotype antibody or antigen-binding fragment thereof of claim 1, wherein the VH region comprises CDR-H1, CDR-H2, and CDR-H3, which CDR-H1, CDR-H2, and CDR-H3 are as set forth in amino acid sequences SEQ ID NOs: 44. 45 and 46; and the VL region comprises CDR-L1, CDR-L2 and CDR-L3, said CDR-L1, CDR-L2 and CDR-L3 being as defined in amino acid sequences SEQ ID NO: 47. 48 and 49, wherein said sequence is obtained from Kabat numbering.
3. The anti-idiotype antibody or antigen-binding fragment thereof of claim 1, wherein
The VL region comprises a sequence identical to SEQ ID NO:40, has at least 90% sequence identity to the VL region amino acid sequence set forth in seq id no; and
the VH region comprises a sequence identical to SEQ ID NO:36, and the VH region amino acid sequences listed in seq id no.
4. The anti-idiotype antibody or antigen-binding fragment thereof of claim 1, wherein
The VL region comprises SEQ ID NO:40, an amino acid sequence of seq id no; and
the VH region comprises SEQ ID NO:36, and the amino acid sequences listed in seq id no.
5. The anti-idiotype antibody or antigen-binding fragment thereof of claim 1, wherein the target antibody or antigen-binding fragment thereof is a single-chain fragment.
6. The anti-idiotype antibody or antigen-binding fragment thereof of claim 5, wherein the target antibody or antigen-binding fragment thereof comprises an scFv.
7. The anti-idiotype antibody or antigen-binding fragment thereof of claim 6, wherein the target antibody or antigen-binding fragment thereof comprises the amino acid sequence of SEQ ID NO:34, and the amino acid sequences listed in seq id no.
8. The anti-idiotype antibody or antigen-binding fragment thereof of claim 6, wherein:
the target antibody or antigen binding fragment is within or contained within an antigen binding domain of an extracellular portion of a Chimeric Antigen Receptor (CAR); and/or
The anti-idiotype antibody or antigen-binding fragment specifically binds to a target antibody or antigen-binding fragment thereof contained within or within the antigen-binding domain of the extracellular portion of the CAR.
9. The anti-idiotype antibody or antigen-binding fragment thereof of claim 8, wherein the antibody or fragment does not bind to an epitope in the spacer domain of the CAR.
10. The anti-idiotype antibody or antigen-binding fragment thereof of claim 8, wherein said anti-idiotype antibody or fragment does not cross-react with another CAR.
11. The anti-idiotype antibody or antigen-binding fragment thereof of claim 8, wherein said anti-idiotype antibody or fragment is an agonist antibody of a CAR comprising said target antibody or antigen-binding fragment.
12. The anti-idiotype antibody or antigen-binding fragment of claim 1, wherein said anti-idiotype antibody or antigen-binding fragment specifically binds to an epitope within or comprising all or part of a Complementarity Determining Region (CDR) of said target antibody or antigen-binding fragment.
13. The anti-idiotype antibody or antigen-binding fragment thereof according to any one of claims 1-4, which is humanized.
14. The anti-idiotype antibody or antigen-binding fragment thereof according to any one of claims 1-4, which is recombinant.
15. The anti-idiotype antibody or antigen-binding fragment thereof of any one of claims 1-4, which is monoclonal.
16. The anti-idiotype antibody or antigen-binding fragment thereof of any one of claims 1-4, which is an antigen-binding fragment.
17. The anti-idiotype antibody or antigen-binding fragment thereof of claim 16, wherein said antigen-binding fragment is selected from the group consisting of Fab fragment, F (ab') 2 Fragments, fab' fragments, fv fragments and single chain variable fragments (scFv).
18. The anti-idiotype antibody or antigen-binding fragment thereof of any one of claims 1-4, comprising at least a portion of an immunoglobulin constant region.
19. The anti-idiotype antibody or antigen-binding fragment thereof of claim 18, wherein at least a portion of the immunoglobulin constant region comprises an Fc region or a portion of an Fc comprising CH2 and CH3 domains.
20. The anti-idiotype antibody or antigen-binding fragment thereof of claim 18, wherein the constant region is derived from human IgG.
21. The anti-idiotype antibody or antigen-binding fragment thereof of claim 20, which is an intact antibody or a full-length antibody.
22. A conjugate comprising the anti-idiotype antibody or antigen-binding fragment of any one of claims 1-21 and a heterologous molecule or moiety that is a label, protein, polypeptide, nucleic acid, or small molecule.
23. The conjugate of claim 22, wherein the heterologous molecule or moiety is a tag, and wherein the label is selected from the group consisting of a fluorescent dye, a fluorescent protein, a radioisotope, a chromophore, a metal ion, a gold particle, a silver particle, a magnetic particle, a polypeptide, an enzyme, streptavidin, biotin, a luminescent compound, or an oligonucleotide.
24. A nucleic acid molecule encoding the heavy and light chains of the anti-idiotype antibody of any one of claims 1-21 or an antigen-binding fragment thereof.
25. A vector comprising the nucleic acid molecule of claim 24.
26. A method of producing an anti-idiotype antibody, or antigen-binding fragment thereof, comprising expressing the heavy and light chains encoded by the nucleic acid of claim 24 in a suitable host cell, and recovering or isolating the antibody.
27. A cell comprising the anti-idiotype antibody or antigen-binding fragment thereof of any one of claims 1-21.
28. A composition comprising the anti-idiotype antibody of any one of claims 1-21, or an antigen-binding fragment thereof, and a pharmaceutically acceptable excipient.
29. A kit comprising the anti-idiotype antibody of any one of claims 1-21, or an antigen-binding fragment thereof, and instructions for use.
30. Use of an anti-idiotype antibody or antigen-binding fragment thereof according to any of claims 1-21 in the manufacture of a medicament for use in a method of detecting a target antibody or antigen-binding fragment thereof, said method comprising:
(a) Contacting a composition comprising a target antibody, or antigen-binding fragment thereof, which is antibody FMC63, with the anti-idiotype antibody of any one of claims 1-21, or antigen-binding fragment thereof, wherein the target antibody comprises the amino acid sequence as set forth in SEQ ID NO:30 and the heavy chain variable region as set forth in SEQ ID NO:31, a light chain variable region shown in seq id no; and
(b) Detecting an anti-idiotype antibody or antigen-binding fragment thereof that binds to said target antibody or antigen-binding fragment thereof.
31. An in vitro or ex vivo method of detecting engineered cells expressing a Chimeric Antigen Receptor (CAR), comprising:
(a) Contacting an engineered cell expressing a Chimeric Antigen Receptor (CAR) comprising a target antibody, or antigen binding fragment thereof, which is antibody FMC63, with the anti-idiotype antibody, or antigen binding fragment thereof, of any one of claims 1-21, wherein the target antibody comprises the amino acid sequence as set forth in SEQ ID NO:30 and the heavy chain variable region as set forth in SEQ ID NO:31, a light chain variable region shown in seq id no; and
(b) Detecting cells bound to said anti-idiotype antibody or antigen-binding fragment thereof,
wherein the method is not used for diagnostic purposes.
32. Use of an anti-idiotype antibody or antigen-binding fragment thereof according to any of claims 1-21 in the manufacture of a medicament for use in a method of detecting a CAR comprising a target antibody or antigen-binding fragment thereof, the method comprising:
(a) Contacting a cell expressing a Chimeric Antigen Receptor (CAR) comprising a target antibody, or antigen-binding fragment thereof, which is antibody FMC63, with the anti-idiotype antibody of any one of claims 1-21, or antigen-binding fragment thereof, wherein the target antibody comprises the amino acid sequence as set forth in SEQ ID NO:30 and the heavy chain variable region as set forth in SEQ ID NO:31, a light chain variable region shown in seq id no; and
(b) Detecting cells that bind to the anti-idiotype antibody or antigen-binding fragment thereof.
33. An in vitro or ex vivo method of selecting cells from a population of cells, comprising:
(a) Contacting a population of cells expressing a Chimeric Antigen Receptor (CAR) comprising a target antibody or a cell that binds to a target antibody with the anti-idiotype antibody or antigen-binding fragment thereof of any one of claims 1-21, wherein the target antibody is antibody FMC63 or antigen-binding fragment thereof, and comprises a polypeptide as set forth in SEQ ID NO:30 and the heavy chain variable region as set forth in SEQ ID NO:31, a light chain variable region shown in seq id no; and
(b) Selecting cells that bind to the anti-idiotype antibody or antigen-binding fragment thereof.
34. An in vitro or ex vivo method of stimulating cells comprising incubating an input composition of cells expressing a Chimeric Antigen Receptor (CAR) comprising a target antibody or antigen binding fragment thereof, said target antibody being FMC63, with the anti-idiotype antibody or antigen binding fragment thereof of any one of claims 1-21, thereby generating an output composition comprising a stimulating cell, wherein said target antibody comprises an amino acid sequence as set forth in SEQ ID NO:30 and the heavy chain variable region as set forth in SEQ ID NO: 31.
35. An in vitro or ex vivo method of producing a cellular composition comprising:
(a) Introducing a nucleic acid molecule encoding a Chimeric Antigen Receptor (CAR) comprising a target antibody that is antibody FMC63 or an antigen binding fragment thereof into a cell, wherein the target antibody comprises a nucleic acid sequence as set forth in SEQ ID NO:30 and the heavy chain variable region as set forth in SEQ ID NO:31, a light chain variable region shown in seq id no; and
(b) Incubating the input composition with the anti-idiotype antibody or antigen-binding fragment thereof of any one of claims 1-21, thereby producing the cell composition.
36. A method of purifying an antibody or antigen-binding fragment thereof, comprising:
(a) Contacting a composition comprising a target antibody, or antigen-binding fragment thereof, which is antibody FMC63, with the anti-idiotype antibody of any one of claims 1-21, or antigen-binding fragment thereof, wherein the target antibody comprises the amino acid sequence as set forth in SEQ ID NO:30 and the heavy chain variable region as set forth in SEQ ID NO:31, a light chain variable region shown in seq id no; and
(b) Isolating a complex comprising the anti-idiotype antibody or antigen-binding fragment thereof.
37. An article of manufacture comprising
(a) The anti-idiotype antibody or antigen-binding fragment of claim 1-21, instructions for using the anti-idiotype antibody to detect FMC63 antibody or antigen-binding fragment thereof or chimeric antigen receptor comprising FMC63 antibody or antigen-binding fragment thereof,
or instructions for selecting or enriching from a population of cells an engineered cell expressing a Chimeric Antigen Receptor (CAR) comprising antibody FMC63 or an antigen binding fragment thereof;
or instructions for stimulating an input composition comprising cells expressing a chimeric antigen receptor comprising an FMC63 antibody or antigen binding fragment thereof; or (b)
(b) A binding agent comprising an extracellular domain of a Chimeric Antigen Receptor (CAR) comprising a target antibody or antigen-binding fragment thereof, said target antibody being antibody FMC63, said extracellular domain or portion thereof comprising said target antibody or antigen-binding fragment thereof, and the anti-idiotype antibody or antigen-binding fragment thereof of any of claims 1-21,
wherein the FMC63 antibody or antigen binding fragment thereof comprises the amino acid sequence as set forth in SEQ ID NO:30 and the heavy chain variable region as set forth in SEQ ID NO: 31.
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